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Zetasizer Nano Series

User Manual

MAN0317 Issue 1.0 June 2003

Malvern Instruments makes every effort to ensure that this document is correct. However,

due to Malvern Instruments policy of continual product development we are unable to

guarantee the accuracy of this, or any other document after the date of publication. 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.

Head office:

Malvern Instruments Ltd.

Enigma Business Park,

Grovewood Road,

Malvern,

Worcestershire. WR14 1XZ

United Kingdom.

Tel + [44] (0)1684-892456

Fax + [44] (0)1684-892789

Windows 2000 and XP are registered trademarks of the Microsoft Corporation.

Zetasizer, NIBS and M3-PALS are registered trademarks of Malvern instruments.

Printed in England

Table of Contents

Part 1 - Operators guide

CHAPTER 1 - Introduction to this manual

Introduction to this manual 1-1 How to use this manual 1-2 Access to the Instrument 1-3 Assumed information 1-4 Where to get help 1-4

CHAPTER 2 - What is the Zetasizer Nano?

Introduction 2-1 What does the Zetasizer Nano do? 2-1 The Zetasizer Nano range 2-1 What is Particle Size, Zeta potential and Molecular weight? 2-3

CONTENTS

CHAPTER 3 - How does the Zetasizer Nano work?

Introduction 3-1 How is a Zetasizer measurement performed? 3-1 What does the Zetasizer consist of ?

- Identifying the Hardware 3-4

- Navigating the Software 3-13

CHAPTER 4 - Making measurements - A tutorial

Introduction 4-1 Quick guide to making a measurement 4-2 Powering up the system 4-2 Sample preparation 4-3 Choosing the correct Cell 4-3 Filling the Cell 4-8 Inserting the Cell 4-11 Making an SOP measurement 4-14 Making a manual measurement 4-16 The Measurement display 4-17 Editing the result 4-23

Zetasizer Nano Series Page 1

CONTENTS

Zetasizer Nano Series

CHAPTER 5 - Records and Reports - Viewing the results

Introduction 5-1 Displaying the results 5-1

CHAPTER 6 - Sample Preparation

Introduction 6-1 Preparing the sample

- Size 6-1

- Molecular weight 6-5

- Zeta potential 6-6

CHAPTER 7 - Maintenance

Introduction 7-1 Cleaning the instrument 7-1 Cleaning the Cells 7-2 Replacing the system fuse 7-4

Part 2 - Supervisors guide

CHAPTER 8 - Security

Introduction 8-1 Initial start-up - set up the administrator 8-2 Enabling security 8-3 User groups 8-3 Users 8-5

CHAPTER 9 - Using SOPs

Introduction 9-1 Creating an SOP 9-2 All SOPs 9-4 Size SOPs 9-10 Molecular weight SOPs 9-18 Zeta potential SOPs 9-22 Trend & Protein melting point SOPs 9-27 Extracting an SOP 9-29 Modifying an SOP 9-29 Distributing an SOP 9-30

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CONTENTS

CHAPTER 10 - Measurement file window - Workspace management

Introduction 10-1 Measurement file window 10-2

CHAPTER 11 - Exporting results

Introduction 11-1 Exporting results 11-1 Creating an export template 11-3

CHAPTER 12 - Creating custom reports

Introduction 12-1 Overview 12-1 Opening a report 12-2 Creating a report 12-2 Laying out a report 12-2 Customising and editing the report 12-4 A finished report 12-11 Viewing the new report 12-12

CHAPTER 13 - Size theory

Introduction 13-1 What is Dynamic Light Scattering? 13-1 Operation of the Zetasizer Nano

- Size measurements 13-6

CHAPTER 14 - Molecular Weight theory

Introduction 14-1 What is Static light scattering? 14-1 The Debye plot 14-4

CHAPTER 15 - Zeta Potential theory

Introduction 15-1 What is Zeta Potential? 15-1 Laser Doppler Velocimetry 15-5 The M3-PALS technique 15-7 Operation of the Zetasizer Nano

- Zeta potential measurements 15-11

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CONTENTS

Zetasizer Nano Series

Part 3 - Appendices

APPENDIX A - Health and Safety

APPENDIX B - Specification

APPENDIX C - Site requirements

Introduction C-1 Environmental conditions C-1 Space required C-2 Power requirements C-3 Additional services C-3 Computer specification C-4 Laser Safety C-4

APPENDIX D - Unpacking instructions

APPENDIX E - Installation

Introduction E-1 Installing the Zetasizer Nano E-1 Changing the computer E-2 Installing the Titrator E-2

APPENDIX F - Regulatory Statements

CE Declaration of Conformity F-1 Federal Communications Commission (FCC) Notice F-2 Canadian Regulatory Information F-3

Page 4 MAN 0317

Part 1 - Operators guide

Introduction to this manual

CHAPTER 1

Introduction to this manual

This manual covers the operation and maintenance of the Zetasizer Nano particle analyser series.

CHAPTER 1

Zetasizer Nano instrument

Nano S (Red badge) ZEN1600 Size measurement particle sizer

Nano S (Green badge) ZEN1500 Size measurement particle sizer

Nano Z (Red badge) ZEN2600 Zeta potential particle sizer

Nano Z (Green badge) ZEN2500 Zeta potential particle sizer

Nano ZS (Red badge) ZEN3600 Size and Zeta potential particle sizer

Nano ZS (Green badge) ZEN3500 Size and Zeta potential particle sizer

Nano S90 (Red badge) ZEN1690 Size measurement particle sizer - 90° optics

Nano S90 (Green badge) ZEN1590 Size measurement particle sizer - 90° optics

Nano ZS90 (Red badge) ZEN3690 Size and Zeta potential particle sizer - 90° optics

Nano ZS90 (Green badge) ZEN3590 Size and Zeta potential particle sizer - 90° optics

Instruments that have a red oval badge fitted to the instrument cover are fitted with a 633nm ‘red’ laser; instruments that have a green badge are fitted with a 532nm ‘green’ laser.

Model number

Description

Build options exist for each of the above instruments, these are described in chapter 2.

Note.The Zetasizer model, Serial number, software and firmware version can

The aim of this manual is to:

be found by left-clicking the Nano icon in the right corner of the status bar.

Identify what the instrument is.

Explain in simple terms how it works.

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How to use this manual

Zetasizer Nano Series

Explain how the instrument should be used to make a measurement.

Identify the user maintenance procedures.

It is important to read the Health and Safety information in appendix A before operating the instrument.

It is recommended this manual is read fully before you start your first measurement, though if more familiar with particle size analysers, jump straight to chapter 4 - “Making measurements - A tutorial”. This chapter gives practical details on making measurements.

The manual is divided into 3 sections.

Part 1 – Operators guide

Contains all the information required for the operator to use the Zetasizer Nano instruments.

Topics covered are: What are the Zetasizer Nano instruments, what are the components of the Zetasizer Nano and what do they do, instructions on using the instrument and the software, basic measurement and maintenance procedures.

Part 2 – Supervisors guide

The Supervisors guide concentrates on the administration and creation of the Zetasizer functionality. It provides a greater insight into the measurement procedures and results expanding the analysis theories.

Topics covered are: security aspects, use of Standard Operating Procedures (SOPs), and organising the measurement files, and discussion on each of the analysis theories used – Zeta potential, Size and Molecular weight.

It is recommended that the supervisor should also read Part 1 – Operators guide

Part 3 - Appendices

Contains supplementary information not necessary for the general operation of the system.

More detail on the Zetasizer software can be found by using the online Help within the software.

Page 1.2 MAN 0317

The Zetasizer Nano measures three different particle characteristics; the text within each chapter has therefore been structured to, detail the instrument functions as applicable to all measurements types, or individually if the function only applies to one, i.e. if concerned only with zeta potential measurements ignore all references to size and molecular weight unless otherwise directed.

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.

Warning!

CHAPTER 1

Removal of the covers by unauthorised personnel will invalidate the warranty of the instrument.

Supervisor

The supervisor is the person responsible for the management and safety of the instrument and its operation. The supervisor is responsible for the training of the operators. They can perform all user maintenance routines identified in chapter 7.

Under no circumstances, should the supervisor remove the main cover of the instrument.

Operator

An operator is a person trained in the use of the system. The operator can perform all user maintenance routines identified in chapter 7, except changing the fuse.

Under no circumstances, should the operator remove the main cover of the instrument.

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Assumed information

Zetasizer Nano Series

Warning!

Failure to follow these guidelines could result in exposure to hazardous voltages and laser radiation.

Naming convention

The Zetasizer Nano will either be referred to in full, as the Zetasizer, or as the ‘instrument’.

The combination of the Zetasizer Nano instrument, the computer and Zetasizer software will be referred to as the "System".

Cells and Cuvettes

Any device for holding and measuring the sample in the instrument will generally be referred to as a “cell”. This includes dip cells and all kinds of cuvettes used (i.e. glass, small volume, disposable) unless a proper description is more appropriate.

Menu commands

Menu commands from the Zetasizer software are referred to in the form main menu-menu item. As an example, the command Configure-New SOP refers

to selecting the New SOP item in the Configure menu. Menu commands are always shown in bold text.

Where to get help

Manual and Help files

The primary source of help for the Zetasizer system is from this manual and the online help system within the software. This manual is designed to give an overview of the system as a whole, while the online help system is designed to give detailed information on the Zetasizer software.

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Each dialogue within the Zetasizer software has a Help button that gives information specific to that dialogue. A secondary type of help exists within the dialogue; this is the Advice button and contains more sample related content.

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 and the front of the cuvette holder).

The build option fitted: a smaller label attached alongside the model and serial number labels identifies any options fitted.

The Zetasizer software version (select Help-About within the software).

Contact the United Kingdom help desk if the local Malvern representative is not available. The direct line to the United Kingdom Helpdesk is +44 (0) 1684

891800. It should be noted that this help line is primarily English speaking.

Remote support

Malvern Instruments offers a remote support service, delivered by an Internet connection. Benefits include fast and efficient fault diagnosis, reducing downtime and costs.

On-line user training is also available, plus software updates. A direct Internet connection LAN must be available to make use of this facility.

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Zetasizer Nano Series

Page 1.6 MAN 0317

What is the Zetasizer Nano?

CHAPTER 2

Introduction

What is the Zetasizer Nano system and what is it supposed to do?

This chapter gives a brief overview of the Zetasizer Nano instrument range: what the Zetasizer Nano does and simple explanations about the measurement technique.

What does the Zetasizer Nano do?

The Zetasizer Nano range of instruments provides the ability to measure three characteristics of particles or molecules in a liquid medium.

These three fundamental parameters are Particle size, Zeta potential and Molecular weight. By using the unique technology within the Zetasizer system these parameters can be measured over a wide range of concentrations. The Zetasizer system also enables determination of the Protein melting point plus the ability to perform Trend measurements.

The Zetasizer range features pre-aligned optics and programmable measurement position plus the precise temperature control necessary for reproducible, repeatable and accurate measurements. In addition facility is included for measurements of other key parameters such as pH and concentration.

The Zetasizer range has been designed with simplicity in mind, so that a minimal amount of user interaction is necessary to achieve excellent results. The use of

Standard Operating Procedures (SOPs) and features such as the Folded capillary cell alleviate the need for constant attention.

The Zetasizer Nano range

There are ten instruments in the Zetasizer Nano particle analyser range: five different models fitted with either a 633nm ‘red’ or 532nm ‘green’ laser. The models and their measurement specifications are described in the table below, with instrument options following.

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Zetasizer Nano Series

Zetasizer

Nano S – 3nm to 10µm –

Nano Z 0.6nm to 6µm – 1000 to 2x10

Nano ZS 0.6nm to 6µm 3nm to 10µm 1000 to 2x10

Nano S90 2nm to 3µm – –

Nano ZS90 2nm to 3µm 3nm to 10µm –

Size range

Size range for Zeta

potential

Size range for

Molecular weight

Daltons

Instrument option

(see below)

>?@

Laser fitted

The Zetasizer Nano series is available with either a 633nm ‘red’ laser or a 532nm ‘green’ laser fitted. The laser fitted is identified by the colour of the oval badge on the cover.

The 633nm laser is least suitable for blue samples.

The 532nm laser is least suitable for red samples.

90° optics

The instruments above with the suffix 90 indicate the optics have a 90° scattering angle. These models have been included in the Zetasizer Nano instrument range to provide continuity with other instruments with ‘classical’ 90° optics.

Instrument options

A range of accessories and options are also available for more advanced measurement strategies.

Narrow band filter

This filter improves the signal for samples that fluoresce at the wavelength of the laser fitted. If fitted an option label will be attached to the front of the cuvette holder.

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Option label Option fitted

ZEN1003 Narrow band filter for all instrument options

Universal ‘Dip’ cell

Used to provide repeatable measurements of non-aqueous samples such as solvents. Also suitable for measurements of valuable aqueous samples where minimal sample quantity is important.

MPT2 Autotitrator

Used to perform sample, pH, conductivity and additive titrations.

What is Particle Size, Zeta potential and Molecular weight?

This section will describe, “basically”, what Particle Size, Zeta potential and Molecular weight are and why they are important. Greater detail on the measurement techniques is given in the theory chapters (13, 14 and 15).

What is Size?

Particle size is the diameter of the sphere that diffuses at the same speed as the particle being measured.

The Zetasizer system determines the size by first measuring the Brownian motion of the particles in a sample using Dynamic Light Scattering (DLS) and then interpreting a size from this using established theories - see chapter 13.

Brownian motion is defined as:

“The random movement of particles in a liquid due to the bombardment

by the molecules that surround them”.

The particles in a liquid move about randomly and their speed of movement is used to determine the size of the particle.

It is known that small particles move quickly in a liquid and large particles move slowly. This movement is carrying on all the time, so if we take two ‘pictures’ of the sample separated by a short interval of time, say 100µS, we can see how much the particle has moved and therefore work out how big it is.

If there has been a minimal movement and the particle positions are very similar, then the particles in the sample will be large; similarly if there has been a large

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CHAPTER 2

Zetasizer Nano Series

amount of movement and the particle positions are quite different, then the particles in the sample are small.

Using this knowledge and the relationship between diffusion speed and size the size can be determined.

mall particles moving quickly

Time

Large particles moving slowly

Time

10 100 1000

Size nm

10 100 1000

Size nm

The above is a very simplistic explanation. A more detailed explanation can be found in chapter 13 - Size theory.

Why do we use it?

Toners and Liquid inks

Image quality, viscosity and the tendency to aggregate and clog ink delivery nozzles are all influenced by particle size. Controlling the size of ink and toner products has a direct effect on image properties, ink permanence and adhesion.

Pigments

Knowledge of Particle size is important in developing stable formulations of pigments. Pigment colour and hue are highly related to particle size, this has applications in determining a pigments properties.

ILL 6722

What is Molecular weight?

The molecular weight of a substance is the weight in atomic mass units (amu)of all the atoms in one molecule of that substance. Mathematically the molecular weight can be calculated from the molecular formula of the substance; it being the sum of the atomic weights of all the atoms making up the molecule.

If we take as an example the molecular formula H molecular weight.

Page 2.4 MAN 0317

O (water) we can work out the

CHAPTER 2

In each molecule of water there are two atoms of Hydrogen (H2) and one atom of Oxygen (O).

Now the atomic weight of hydrogen is 1.008 amu and that of oxygen is

15.999.

Therefore the molecular weight of water is 18.015 i.e.(1.008 x 2)+15.999.

1.0081.008

15.999

1.0081.008

18.015

This is a calculation using a known molecular formula and applying the values from the periodic table.

With the Zetasizer Nano series of instruments the molecular weight can now be determined by use of Static Light Scattering (SLS) measurement techniques.

This technique will be more explained in chapter 14 - Molecular weight theory.

Note

Malvern uses Daltons to identify the molecular weight.

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 being the chemistry standard term for ‘1 molecular weight’, e.g. one mole of water is 18.015g).

In an application, knowing the molecular weight of polymer compounds will aid in determining many of their physical characteristics such as density, flexibility and strength.

ILL 6721

What is Zeta potential and Electrophoresis?

Most liquids contain Ions; these can be negatively and positively charged atoms called Cations and Anions respectively. When a charged particle is suspended in a liquid ions of an opposite charge will be attracted to the surface of the suspended particle.

i.e. - a negatively charged sample attracts positive ions from the liquid and conversly a positive charged sample attracts negative ions from the liquid.

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Zetasizer Nano Series

Ions close to the surface of the particle, will be strongly bound while ions that are further away will be loosely bound forming what is called a Diffuse layer. Within the diffuse layer there is a notional boundary and any ions within this boundary will move with the particle when it moves in the liquid; but any ions

outside the boundary will stay where they are – this boundary is called the Slipping plane.

Diffuse layer

Zeta

Ions loosely attached

otential

Negatively charged particle

Ions strongly

bound to particle

Slipping plane

+ +

ILL 6747

A potential exists between the particle surface and the dispersing liquid which varies according to the distance from the particle surface – this potential at the slipping plane is called the Zeta potential.

Zeta potential is measured using a combination of the measurement techniques: Electrophoresis and Laser Doppler Velocimetry, sometimes called Laser Doppler Electrophoresis. This method measures how fast a particle moves in a liquid when an electrical field is applied – i.e. its velocity.

Once we know the velocity of the particle and the electrical field applied we can, by using two other known constants of the sample – viscosity and dielectric constant, work out the zeta potential.

This technique will be further explained in chapter 15 - Zeta theory.

Why do we use it?

The zeta potential of the sample will determine whether the particles within a liquid will tend to flocculate (stick together) or not.

Knowledge of zeta potential is therefore useful in many industries such as:

Ceramics: A high zeta potential is required to ensure the ceramic particles are densely packed. This gives added strength to the end product.

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CHAPTER 2

Waste water treatment.

The flocculation state of waste water is altered by changes in pH, the addition of chemical flocculants, such as charged polymers, and the presence of aluminium chloride or other highly charged salts. Measurement of zeta potential in combination with these parameters is fundamental in the development and maintenance of optimized water treatment protocols.

Emulsions. Zeta potential is used to study the chemistry involved in determining whether or not an emulsion will remain stable in the environment where it will be used.

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Zetasizer Nano Series

Page 2.8 MAN 0317

How does the Zetasizer

Nano work?

CHAPTER 3

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 what is involved in making a measurement; what the major components of the system are and how the software performs the task. This is followed by two sections identifying the major hardware components and the software used.

The complete measurement process for size, zeta potential and molecular weight measurements will be described in later chapters.

How is a Zetasizer measurement performed?

A typical system, shown above, 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..

1 4 3 2

Zetasizer Nano Series Page 3.1

ILL6744

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Zetasizer Nano Series

The software is used to control the measurement of the sample, there are two basic ways to make a measurement:

SOP measurements. A Standard Operating Procedure (SOP) is like a template that pre-defines all the measurement settings. This ensures that measurements made on the same type of sample are made in a consistent way. SOPs are ideal if the same type of sample is regularly measured, inputting the same parameters each time a measurement is made is tedious and runs the risk of making errors.

SOPs can be created or modified as required.

To perform an SOP measurement, select Measure-Start SOP from the menu bar and select an SOP to use. With an SOP chosen the Measurement display will appear (below). The measurement will begin by pressing the Start ($) button.

Manual Measurement. A manual measurement is where all the measure ment parameters are set immediately before the measurement is performed. This is ideal if measuring many different types of sample, or experimenting with the measurement parameters.

To perform a manual measurement, select Measure-Manual from the menu bar. A manual measurement dialogue window will appear where the measurement settings can be chosen, and if required saved as an SOP. Once chosen the measurement can begin by simply pressing the Start ($) button on the Measurement display.

Page 3.2 MAN 0317

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CHAPTER 3

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-.

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.

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CHAPTER 3

What does the Zetasizer consist of ?

Zetasizer Nano Series

- Identifying the Hardware

The diagram below shows a typical system with its key modules of 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.

The software controls the Zetasizer and any accessories used and analyses the data from the instrument to give either the size, molecular weight or zeta potential for the sample measured.

The Zetasizer Nano instrument

6 3

ILL 6739

ILL 4022

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CHAPTER 3

Optical unit

Positioned on the cover are two labels - one to identify the instrument and another to identify the instrument model; refer to the identification table in chapter 2.

Rear Panel

The rear panel provides all the connections. These are identified below.

D GF

ILL 6737

Power input socket

Mains power input socket for the instrument.

Fuse holder

Fuse for the instrument. Details on changing the fuses can be found in chapter 7 Maintenance.

Power switch

The on/off power switch for the instrument. On switch on the “status indicator”-around the Cell access button.will illuminate.

Computer connection

The USB cable from the computer is connected here.

Accessory connections

Two types of connection are available.

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Zetasizer Nano Series

CAN Ports+and

Use these ports to connect any Malvern supplied accessory that requires a CAN connection (Controller Area Network). Consult the respective accessory manual for details.

RS232 (I0I0I) Ports+and

Use these ports to connect any Malvern supplied accessory that requires an RS232 connection. Again consult the respective accessory manual for details.

Control of any accessory is done via Zetasizer software.

Cooling fans

In conjunction with ventilation slots underneath the instrument, the fans provide cooling to the internal components of the Zetasizer.

Do not obstruct the ventilation slots underneath the instrument, nor the fans on the rear panel.

Serial number and Model number label

Identifies the actual Zetasizer Nano model and its serial number. Please quote all numbers in any correspondence with Malvern Instruments.

The option label indicates any instrument options fitted into the Zetasizer.

For an explanation refer to the identification table in chapter 2.

Accessory output

A 12v output supply is provided on the rear panel to connect to any Malvern supplied accessory that requires an external voltage source. Consult the respective accessory manual for details.

Only connect Malvern approved accessories.

Green laser PSU input

If the instrument uses a 532nm ‘green’ laser, connect the laser PSU to this connection.

Purge connection

If a Nitrogen supply is used the instrument must be located in a well ventilated environment. The purge supply expels 50ml of Nitrogen a minute. Ensure the supply is turned off when not in use.

If measuring samples at low temperatures there is a risk of condensation occurring on the cell; this occurs when the measurement temperature is less than the ‘dew point’ of the ambient air surrounding the cell. This is particularly prevalent in humid climates. If it is suspected that this may be a problem then the

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CHAPTER 3

purge inlet port can be used to connect an air or nitrogen supply. This will remove any moisture in the air immediately surrounding the cell and prevent condensation.

If using the purge inlet connector then the air or nitrogen supply must conform to the following specification:

Compressed air to DIN 8573-1

Oil = Class 1

Water = Class 3

Particulate = Class 3

Pressure = 200 ± 20 kPa g

On entry into the Zetasizer the flow rate of the purge air is controlled by a built in regulator.

Caution

It is important that the purge air line supply conforms to the above specification. Failure to meet this specification may result in permanent damage and invalidation of the warranty.

Status indicator

The status indicator is an illuminated ring (or bezel) positioned around the Cell access button., and shows the operational state of the instrument.

Indicator colour and state

Amber - Flashing Shows the start-up initialisation routine is running.

Amber Shows that the instrument is at “Standby”.

Green

Green - Flashing Occurs when the instrument is performing a measurement.

Zetasizer Nano Series Page 3.7

Function

The instrument is functioning correctly but is either not connected to the computer or the software has not been started.

Indicates the instrument is functioning correctly and is ready to

start a measurement.

CHAPTER 3

Zetasizer Nano Series

Red Indicates if the instrument has detected an error. The

measurement will be stopped.

Amber is a combination of red and green lights.

Cell access button

Positioned in the middle of the Status indicator-, pressing the button will open the cell area lid.

Cell area

Warning

The system is capable of heating the cell to high temperatures. Care should be taken when removing the cells if a measurement has been performed at high temperatures. It is recommended that the cell area is allowed to cool before removing the cell. A warning triangle is provided in the cell basin.

The cell area is where all cells are inserted to undertake a measurement. The cell area is completely self enclosed and controls the sample temperature over the range 2°C to 90°C.

If the lid is opened with the cell area temperature above 50°C the instrument will ‘beep’ twice every few seconds to warn of high temperatures.

Note.When the Zetasizer is initially switched on the cell area will be driven to

a “default” temperature of 25°C. This will also happen if the software is closed, but the instrument is not switched off.

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Other important features of the cell area are described below.

CHAPTER 3

Cell area lid

On pressing the cell access button the lid will slowly raise allowing access to the cell holder. On opening two safety interlocks are activated.

Laser safety interlock. This interlock prevents any laser light from entering into the cell area.

Electrode voltage interlock. This interlock immediately turns off any voltage to the cell electrodes

To close the lid push it down until it locks. No measurements can be performed unless the lid is fully closed.

Electrodes

The electrodes perform two functions.

Provide voltage for zeta potential measurements. Voltage is immediately turned off when the cell lid is opened.

Note.The maximum working voltage on the electrodes is ±160v

(Measurement category I).

ILL 4022

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Zetasizer Nano Series

Identifies the zeta cell fitted.

Depending upon the measurement to be performed, either a size or zeta potential cell will be required.

When a zeta potential cell is inserted, the electrodes positioned on either side of the cell holder will enable the type of cell to be detected automatically. During the measurement the electrodes supply the voltage necessary to perform electrophoresis.

If a size cell is inserted no electrode contact is made and the Zetasizer assumes a size cell is fitted.

Note.The software will indicate if the wrong cell has been inserted for the

Access for flow cell tubes

A channel incorporated into the cell area allows sample tubes to be connected to the measurement cell. This facility is used with the MPT2 Autotitrator accessory.

The channel includes a pinch valve to hold the tubes in place and stop sample movement during the measurement. To fit the tubes slide them into the channel until clamped by the pinch valve.

measurement.

Cell basin

The cell basin is made of an insulating material which provides protection from the heated cell holder and in conjunction with the Thermal capBgives temperature stability when heating and cooling the sample.

A warning label indicates that high temperatures may exist in the cell area.

Thermal cap

Warning

With the thermal cap removed both the metal lining of the the cap and the top of the cell holder will be exposed. Care should be taken when removing the cells after a measurement has been performed at high temperatures. It is recommended that the cell area is allowed to cool before removing the cell. A warning triangle is provided on the top of the cap.

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The thermal cap gives increased temperature stability for size measurements when heating and cooling the sample during size measurements. This is important when measuring at either end of the temperature specification. The thermal cap is placed over the cell to ensure the temperature requested is reached.

A parking position is provided in the cuvette holder to store the thermal cap.

Note.The thermal cap is not compatible with the folded capillary cell.

Drain port

In case of spillage within the cell area, there is a drain incorporated into the base of the cell holder.

Any spillage will exit onto the bench area underneath the Zetasizer.

Drain channel

Likewise in case of spillage on the cover a drainage channel is provided around the outside of the cell area, this is hidden from view under the main cover. Any spillage will flow along the drain into a hole positioned at the back of the cell area.

Any spillage will exit onto the bench area underneath the Zetasizer.

Cuvette holder

The cuvette holder is for storing the cells before and after use. The cuvette holder swings out from under the instrument and up to 12 cuvettes can be stored.

ILL 6735

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Zetasizer Nano Series

Caution

Ensure the thermal cap is lowered and all cuvettes have been removed before swinging the holder back under the instrument base.

The two trays that hold the cuvettes can be removed for cleaning.

The cuvette holder provides a place to store the thermal cap during changeover of cells. The cap is released by raising the cap and lifting off the cap post.

Similarly storage is provided for the two cell “thermal contact plates” used with the folded capillary cell. These work in the same manner as the thermal

capBabove and provide thermal accuracy and stability. Place these in the holder

to the left of the tray.

The cuvette holder includes a serial number, model number and option labels These identify the instrument and should be quoted in any correspondence with Malvern Instruments. Refer to the identification table in chapter 2.

Note.The Zetasizer Nano model, serial number, software and firmware

Cells and Cuvettes

A range of cells and cuvettes are available to use with the Zetasizer instrument. Full details are given in the making measurement chapters, but briefly the following cells can be used.

version can be found by left-clicking the Nano icon in the right corner of the status bar.

Cell Application

Disposable “polystyrene” cuvettes – Standard and Small volume

Quartz glass cuvettes – Square – Standard, Low and ultra-low volume, flow

Folded capillary cell For Zeta potential, Size and Molecular weight

Universal ‘Dip’ cell For Zeta potential

For Size and Zeta potential (with dip-cell)

For Size, Molecular weight and Zeta potential (with dip-cell)

Page 3.12 MAN 0317

Navigating the Software

The Malvern Zetasizer software controls the system during a measurement and then processes the measurement data to produce either a size, zeta potential or molecular weight result. It displays the results and allows reports to be printed.

There are two modules that are incorporated into the standard Zetasizer software.

The main Zetasizer application which is described below

A secondary module known as Report Designer which enables custom re ports to be created to display the results. The features of Report Designer are detailed in chapter 12.

The next section describes the key features of the main application.

Quick guide to the Zetasizer Nano software application

A typical screen is shown below. The features and their function are described in the following sections.

CHAPTER 3

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Zetasizer Nano Series

+ 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.

Items that are shown in grey indicate that they are not available. Grey items indicate that some security setting has been activated, or that the item is not relevant to the system connected.

The Menus available are shown below, together with a brief overview.

File Menu

The File menu is used to Open… or create a New measurement file. The measurement file

is where all the measurement records (results) will be stored. Select Save as… to store a measurement file under a different file name.

Once a measurement file has been created, use Export ... to export the measurement details to other software packages such as Excel or Wordpad.

Select Batch print... to simultaneously print a number of measurement records.

Create PDF... is only enabled if the 21 CFR part 11 feature key has been installed.

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.

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Edit Menu

Allows movement and manipulation of records in the Measurement file window(s). Records can be cut, copied, pasted and deleted into their own or other measurement files.

Edit result... allows an existing measurement record to be reanalysed using different dispersant and particle properties. Comments on the editing reason added. The edited measurement will then be added to the bottom of the Records view listing.

To view the settings for any particular measurement record, select the record

and then Extract SOP.... The SOP

dialogues will appear showing the original measurement settings. These can then be saved as an SOP so measurements can be made again with the same settings. This is useful if the parameters of the measurement record are not already saved in the SOP directory.

CHAPTER 3

Note.The Edit menu will appear when the right mouse button is pressed

anywhere in the measurement file window.

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Zetasizer Nano Series

View Menu

This selects which reports will be shown in the measurement file window and which Toolbars are to be displayed.

Workspaces will select the workspace toolbar, and which measurement workspace is to be displayed.

The reports available are those selected when creating the workspace with the Configure-Workspace dialogues. The reports available will change to match the workspace chosen.

Configure Menu

Use this menu to create or edit the measurement settings in an SOP prior to performing the measurement.

New SOP... opens the SOP creation wizard, while Existing SOP... allows a previously created SOP to be altered.

Select Status bar to toggle the status bar from being displayed or not.

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Data Export Templates...

opens a dialogue that defines the parameters and the format in which the record data will be exported. Once a template is created the measurement data can be exported to other software packages such as Excel or Wordpad, by using

File-Export data.

Line styles... allows the

colour and style of lines to be changed in the report graphs.

The reports available, and the parameters shown in the Records view tab, are those selected when creating the measurement file workspace with the Workspace... dialogues. The dialogues enable workspaces to be deleted and Added,or previously created ones to be displayed.

CHAPTER 3

Measure Menu

Select this menu when ready to perform a measurement.

There is a choice of using an existing measurement SOP or manually setting up the measurement and sample details.

Once the measurement details have been entered or an SOP has been chosen, the Measurement display will appear.

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Zetasizer Nano Series

Tools Menu

The operation of Report Designer is explained in

chapter 12.

Serial port... allows the USB communications port to which the instrument is connected to be chosen. All instruments connected will be shown in the related dialogue.

Engineering allows Malvern authorised service engineers to perform maintenance tasks. The engineering screens are password protected.

Security Menu

To prevent unauthorised changes, the Malvern software can be configured to limit each users access to various functions - e.g. modifying an SOP. Users are assigned operating permissions that allow, or restrict, access; this will be fully explained in chapter

Page 3.18 MAN 0317

Window Menu

Use this menu to alter the view characteristics of any measurement file windows that are open - i.e. minimise, tile, and cascade the measurement file windows as required.

Select Window-Windows... to open the view dialogue.

Help Menu

Help Topics... give access to the help files.

Tutorials... provides a list of easy to follow

tutorials describing various aspects of the system.

CHAPTER 3

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.

About… gives details on the software version installed. It is helpful to quote this if contacting Malvern Instruments.

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.

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To identify each tools function, move the cursor over a tool - a tool tip will be displayed under each tool with a short description of its action displayed in the status bar.

As with the menu bar, if a tool is not available it will be shown “greyed out”.

The content and appearance of the toolbar can be changed using the View-Toolbars-Customise option.

Measurement file window.

The measurement file window displays all the information for ‘one’ measurement file. More than one measurement file window can be displayed at a time. The contents of the window will change when a Record or Report tab is selected.

Manipulation of the measurement file windows is described in chapter 10.

Measurement file workspace

When performing zeta potential measurements, it may be unnecessary to see parameters associated with size measurements in the measurement file window. A measurement file workspace called zeta potential is available that displays only parameters associated with zeta potential measurements.

Workspaces allow configurable selection of record view parameters and reports that are only relevant to a particular measurement type. Similarly a user can create a personalised workspace so that only parameters and reports relevant to themselves will be shown.

Record and Report tabs.

Measurement records are viewed with the Records view tab; this gives a listing of all the measurement records in any measurement file. The Records view tab is always shown as the prominent report tab when a new measurement file is opened.

The parameters shown are selected by the Record View parameters tab in the Workspace dialogues.

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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.

Details on interpreting the reports can be found in chapter 6.

Title bar.

The title bar displays the software name and the file name of the currently selected measurement file.

Measurement display

When a measurement is being performed a measurement display (below) will appear showing the progress of the measurement. The screen display shown depends on the type of measurement being performed and the view tab selected.

Status bar and 21 CFR 11 icon

The status bar gives an indication of the instrument’s current operating state and gives an extended description of the tool icons. If required, use the View menu to disable.

Double-clicking the Nano icon will display the Zetasizer Nano model, serial number, software and firmware version of the instrument (only if the instrument is connected and switched on).

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Zetasizer Nano Series

If the 21 CFR part 11 feature key is installed, the “21 CFR 11” icon will appear in yellow on the right of the status bar. Double-clicking the icon will display the feature key number. Please note that the 21 CFR part 11 option is not detailed in this manual.

Malvern defined features

Within the software, various parameters, settings or reports will have either a small 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.

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Making measurements -

A tutorial

CHAPTER 4

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. The rest of the chapter will go through the same steps but in more detail.

Once a measurement has been completed, the result can be edited to check the effects if one of the measurement parameters was altered - refer to Editing the result at the end of the chapter.

Manual and SOP measurements

It was mentioned in Chapter 3 that there are two basic measurement methods: Manual measurements and Standard Operating Procedure (SOP) measurements. It is important to understand and consider these methods before proceeding.

A Manual measurement is basically a one-off measurement where all the measurement parameters are set up immediately prior to the measurement. This is ideal if measuring many different types of sample, or experimenting with the measurement parameters.

An SOP measurement uses pre-set parameters (that have previously been defined) to ensure that measurements made on the same type of sample are made in a consistent way; this is useful in quality control environments. SOPs are also ideal if measuring the same sample in slightly different ways; having to type a majority of identical parameters each time a measurement is made is tedious and runs the risk of making errors in the settings. In stead, alter an existing SOP and just change the required parameters.

CHAPTER 4

The sections that follow, “Quick guide to making a measurement” etc, will focus on SOP measurements. Chapter 9 will give details on creating and managing your own SOPs.

Note that most of the settings and dialogues used for a manual measurement are the same as those used in an SOP measurement.

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CHAPTER 4

Quick guide to making a measurement

Zetasizer Nano Series

This section will give a brief overview of the measurement process using an SOP. More information on each stage can be found later in this chapter.

Close the lid and Turn on the instrument and wait 30 minutes for the la ser to stabilise.

Start the Zetasizer software.

Prepare the sample following the sample preparation guidelines.

Choose the cell(s) appropriate for the sample and measurement type.

Fill the cell(s) with the prepared sample.

Make an SOP measurement.

If necessary Open or create a new measurement file.

Select Measure-Start SOP from the Zetasizer software. Select the SOP required and select Open.

Follow any onscreen instructions that appear.

The Measurement display will now be shown.

When requested, insert the cell into the instrument and wait for the temperature to stabilise.

Click Start ($). The measurement will be made and the results displayed and saved to the open measurement file.

Powering up the system

To power up the system, Turn on the instrument and then Start the software.

Turning on the instrument

On switch on an initialisation routine is performed that checks the instrument is functioning correctly.

Close the lid and turn on the optical unit, switch on the power at the power socket and turn the power switch at the rear of the unit on.

A “beep” will occur to indicate the instrument has been turned on and the initialisation routine will begin, followed by a second “beep” once the instrument has finished the routine. Two further beeps will be heard to indicate the instrument has reached the “default” temperature of 25°C.

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CHAPTER 4

Note

Important! All laser based measuring instruments should be left powered up for approximately 30 minutes before measurements are made. This is to prevent any thermal equilibration problems affecting the measurement results.

Starting the Zetasizer Nano software

Double click on the icon to start the software.

If the desktop icon is not available, select

Start-Programs-Malvern Instruments-DTS-DTS

to start the program.

Sample preparation

The process of making a measurement is very simple - insert the sample into the instrument and then use the software to run either an SOP or manual measurement. However, the preparation of the sample before it is inserted into the instrument is paramount.

Please refer to Chapter 6 for sample preparation guidelines for the different measurement types.

Choosing the correct Cell

Malvern offers a range of cells for performing measurements with the Zetasizer system. Choice of cell is dependent upon the type of measurement being performed and the sample that will be measured.

The choices for each measurement type are outlined below with some discussion on their use.

General advice

Generally, for “easy to perform” measurements, such as with samples that scatter a reasonable amount of light (latex with 0.01% mass or higher, high scattering intensity etc.) the disposable polystyrene cuvettes can be used.

Disposable cuvettes are easily scratched though and should never be used more than once.

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Disposable polystyrene cuvettes are not resistant to organic solvents, thus non-water based samples should generally be measured in glass or quartz type cuvettes.

The optical quality of the cells is vitally important when performing molecular weight and protein measurements, therefore glass or quartz type cuvettes should be used to ensure the optimum signal is achieved.

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.

Caution!

Due to the risk of melting, polystyrene cuvettes must not be used for measurements above 50°C.

Size measurements

“Size & Zeta” Folded Capillary cell (DTS1060)

Typical solvent Water, Water/alcohol Water, Water/ethanol

Optical quality Good to very good Good to very good

Minimum Sample volume

Advantages Low cost.

Disadvantages Not resistant to organic solvents

0.75ml 1ml

Single use disposable (no cleaning)

Use with MPT2 Autotitrator

No sample cross-contamination

Fast sample change over

Unsuitable for use at high temperatures (above 70°C)

Disposable polystyrene (DTS0012)

Low cost

Single use disposable (no cleaning)

Not resistant to organic solvents

Unsuitable for use at high temperatures (above 50°C)

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Disposable low volume polystyrene (DTS0112)

Typical solvent Water, Water/alcohol Water, most organic and inorganic

Optical quality Good to very good Excellent

Minimum Sample volume

Advantages Low cost

Disadvantages Requires careful filling to avoid

375µl (100µl when using insert) 1ml

Low volume

Single use disposable (no cleaning)

bubbles

Not resistant to organic solvents

Unsuitable for use at high temperatures. (above 50°C)

Glass - square aperture (PCS1115)

Glass - round aperture (PCS8501)

solvents

Highest optical quality

Can use nearly any dispersant

Requires cleaning after measurement

Low volume quartz (DTS2145)

Typical solvent Water, most organic and inorganic

solvents

Optical quality Excellent Excellent

Minimum Sample volume

Advantages Highest optical quality

Disadvantages Requires cleaning after

Zetasizer Nano Series Page 4.5

1ml 20µl

Can use nearly any dispersant

Reusable

measurement

Water, most organic and inorganic solvents

Highest optical quality

Can use nearly any dispersant

Low sample volume

Requires cleaning after measurement

Requires careful filling to avoid bubbles

CHAPTER 4

Zetasizer Nano Series

Low volume Glass flow cuvette (ZEN0023)

Typical solvent Water, most organic and inorganic

solvents

Optical quality Excellent

Minimum Sample volume

Advantages Highest optical quality

Disadvantages Requires cleaning after

75µl plus tubing

Can use nearly any solvent (tubing dependent)

Use with Autotitrator

measurement

With manual use requires careful filling to avoid bubbles

Molecular weight measurements

Glass - round aperture (PCS8501)

Typical solvent Water, most organic and inorganic

solvents

Optical quality Excellent Excellent

Glass - square aperture

(PCS1115)

Water, most organic and inorganic solvents

Minimum Sample volume

Advantages Highest optical quality

Disadvantages Requires cleaning after

Page 4.6 MAN 0317

1ml 1ml

Highest optical quality

Can use nearly any dispersant

Reusable

measurement

Can use nearly any dispersant

Reusable

Requires cleaning after measurement

CHAPTER 4

Zeta potential measurements

“Size & Zeta potential” Folded Capillary cell (DTS1060)

Description This is a maintenance-free capillary cell primarily designed for zeta

potential measurements.

It has been designed to be used for a single measurement or series of measurements, then discarded rather than cleaned. This removes the chances of cross-contamination. The cell can be inserted either way round.

The cell provides a low-cost alternative to previous reusable quartz capillary cells.

The stoppers can be replaced with ‘Luer’ connectors to provide leak-free connection to the optional MPT2 Autotitrator.

Size measurements can also be performed without having to

ILL 6733

remove and reposition the cell.

Sample details can be written on the textured area on the side of the cell with a permanent pen.

Application The cell is used for measurements of aqueous based samples

Typical solvent Water, Water/alcohol

Optical quality Good to very good

Minimum

0.75ml

Sample volume

Advantages Low cost

Single use disposable (no cleaning)

Use with Autotitrator

No sample cross-contamination

Fast sample change over

Disadvantages Not resistant to organic solvents

Unsuitable for use at high temperatures (above 70°C)

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Universal ‘Dip’ cell (ZEN1002)

Description The Universal ‘Dip’ cell is used to provide a method to measure the

zeta potential of both aqueous and non-aqueous samples - A number of samples can be prepared and the Dip cell inserted to measure each one in turn.

For aqueous samples the dip cell can be used in conjunction with the disposable polystyrene (DTS0012), and for non-aqueous samples use the reusable Glass - square aperture (PCS1115). These cells are described above.

ILL 6764

Application The ‘Dip’ cell can be used for measurements of aqueous and

non-aqueous based samples.

To avoid cross-contamination between aqueous and non-aqueous sample, the dip cells are fitted with colour-coded caps and the ability to be identified as such by the software. It is suggested that the blue capped cell is used for aqueous samples, and the green capped cell used for non-aqueous.

Filling the Cell

When filling the cell there are several actions to consider; some that applies to all cells and other actions that are only applicable to the measurement type and the cell chosen.

General advice

Only clean cells should be used. All size and zeta potential cells should be rinsed/cleaned with filtered dis persant before use. All molecular weight cells should be rinsed/cleaned with the filtered stan dard(i.e. Toluene) or solvent before use.

The cell should be filled slowly to avoid air bubbles from being created. Ultrasonication can be used to remove air bubbles - but only if the sample is suitable for use with ultrasonics.

If using syringe filters for the dispersant, never use the first few drops from the syringe, in case there are any residual dust particles in the filter that may contaminate the dispersant.

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Size and Molecular weight measurements

A minimum sample volume must be provided. However, this minimum volume depends on the actual cell type and it is easier to ensure a certain depth of the sample in the cell. This minimum is 10mm from the bottom of the cell (the measurement is made 8mm from the bottom of the cell).

Min. 15mm

Min. 10mm

Do not overfill the cell, about 15mm maximum, as this can produce thermal gradiants within the sample that will reduce the accuracy of the temperature control.

Zeta potential measurements

The two cells used for zeta measurements are the folded capillary cell and the dip cell; the dip cell will use square cuvettes to hold the sample. Though filling either cell is a simple task, there are a number of precautions to be aware of.

Universal ‘Dip’ cell

With the insertion of the dip cell the sample will be displaced upwards within the cuvette. If too much sample is placed into the cuvette prior to insertion of the dip cell there is a risk that the cuvette will overflow.

ILL 6731

To ensure a minimum sample volume is provided for the sample to be measured, but protect against overfilling we recommend the cuvette is filled to a depth of

Min. 10mm

Min. 7mm

ILL 6730

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Zetasizer Nano Series

between 7mm and 10mm (before the dip cell is inserted). The minimum level relates to approximately 0.7ml of sample.

Do not overfill the cell; as well as overflowing the cuvette once the dip cell is inserted, this can also produce thermal gradiants within the sample that will reduce the accuracy of the temperature control.

If necessary tap the cell lightly to dislodge any bubbles that may be caught between the electrodes.

Folded capillary cell

Fill the cell as described below

Prepare the sample in a syringe of at least 1ml capacity.

Place the sample syringe into one of the sample ports.

Slowly inject the sample through the cell+, checking that all air bubbles are removed. If a bubbles forms under the sample port, pull the syringe plunger back to draw the bubble into the syringe body and then reinject.

Once sample starts to emerge from the second sample port, insert a

stopper,.

Remove the syringe and replace with a second stopper-.

No bubbles should be seen within the clear capillary area of the cell. If necessary tap the cell lightly to dis lodge them. Check that the cell electrodes are still completely cov ered.

Remove any liquid that may have spilt onto the electrodes.

ILL 6734

Note

Page 4.10 MAN 0317

The stoppers must be fitted before a measurement is performed.

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 next section. When and how the cell is inserted will depend on the application, and the measurement choices selected.

Size and Molecular weight measurements

CHAPTER 4

Small triangle

towards button

Open the cell area lid by pushing the button in front of the lid.

Push the cell into the cell holder until it stops. Some cells have opaque surfaces

as well as polished optical surfaces. A polished optical surface must be facing the front of the instrument (towards the button). 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.

If a flowcell is used, insert the sample tubes into the threaded inserts and screw into the top of the flowcell, and then push both tubes down into the pinch valve on the side of the cell area.

To Autotitrator

Flowcell connections

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Zetasizer Nano Series

Place the thermal cap over the cell; Do not fit if using the flowcell.

Close the cell area lid.

Zeta potential measurements

The two cells used for zeta potential measurements are the folded capillary cell and the dip cell; the dip cell needs to be used with a cuvette with a square aperture. Both cell types involve slightly different insertion routines.

Note.The electrode contacts on each cell, as well as applying the

Inserting the Folded capillary cell

Place a thermal contact plate into the recess on either side of the folded

capillary cell. The plates provide increased temperature stability.

measurement voltage, provide identification to the software of which zeta potential cell is fitted.

Open the cell area lid by pushing the button in front of the lid.

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Hold the cell near the top, away from the lower measurement area, and push

into the cell holder until it stops. The cell can be inserted either way round.

Close the cell area lid.

Inserting the Universal ‘Dip’ cell

Insertion of the dip cell, with cuvette, into the cell holder is the same procedure as above, but first the dip cell must be placed into the sample cuvette.

Place the dip cell fully into the cuvette. Some cells have opaque surfaces as well as polished optical surfaces. Ensure the small triangle at the top of the cell faces the front of the instrument. Check that the sample does not overflow the cuvette when the dip cell is fully inserted.

Stop

Sto

Holding the base of the dip cell cap and the top of the cuvette simultaneously, push the cell into the cell holder until it stops - a ‘stop’ on the dip cell must rest on the top of the cell holder.

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CHAPTER 4

Making an SOP measurement

Zetasizer Nano Series

Caution.

When removing the dip cell, and cuvette, ensure that both the dip cell cap and cuvette are held when the cell is withdrawn. If only the dip cell is removed there is a risk of drops of sample falling from the bottom of the dip cell onto the instrument and surrounding area. This is especially important with solvent based samples.

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. Now all that remains is to open or create a measurement file, open the required SOP, place the filled cuvette into the instrument and finally to press the Start ($) button.

This process is outlined below. Chapter 9 gives all the detail required to create new SOPs.

Opening or creating a Measurement File.

Each time a measurement is made, the measurement data will be saved to a measurement file. How the measurement files are managed is down to preference. As an example:

One measurement file may be used for all the measurement records (not recommended).

Separate files are used for each type of sample i.e. one for titanium dioxide and one for carbon black.

A separate measurement file is used for each week or month.

A separate measurement file is used for each user.

Note.If more than one measurement file window is open, the measurement

record will be saved to the measurement file currently active. When the software starts it will automatically open the last measurement file used.

Page 4.14 MAN 0317

To open an existing measurement file:

Select File-Open.

A dialogue will appear allowing selection of a measurement file.

Select Open.

To create a new measurement file:

Select File-New.

A dialogue will appear allowing the new measurement file to be named and specify where it will be saved.

Select Save.

CHAPTER 4

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. Select the SOP that will be used and select Open.IfanSOP has not been specified for the sample, read chapter 9 for details on how to create one.

Pre-measurement instructions may appear to advise of any actions that need to be performed before the measurement can proceed.

This may be followed by a Labels dialogue, allowing the measurement to be named (displayed as the Sample name in the records view). This dialogue also allows any other information about the measurement to be entered in the General notes box, such as a batch number etc. Once the measurement record has been named and any comments added, select the OK button.

The Measurement display, discussed below, will now appear.

Note.It may be that the SOP was not configured to automatically show the

Labels dialogue. If the dialogue does not appear, but is required, select the Settings button in the measurement display.

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CHAPTER 4

Making a manual measurement

Zetasizer Nano Series

Follow the instructions on the status line of the measurement display - i.e. Insert the Cell (described above) and press the Start ($) button to start the

measurement.

The progress of the measurement can be viewed in the measurement display. The measurement may take anything from 2 minutes to over an hour per measurement, depending on the settings within the SOP.

Once the measurement sequence (below) is complete the measurement display can be closed and the new record will be shown in the measurement file window. The results can now be viewed - see Displaying the results in chapter 5.

Making a manual measurement is essentially the same as making an SOP measurement, except, where in an SOP measurement all the measurement options are pre-specified, it will be necessary to set them immediately. All the dialogues are available at once in a tabbed format.

Follow the measurement procedures described above. Instead of starting an SOP, select Measure-Manual. This will open the Manual measurement settings dialogue allowing any measurement types to be chosen and the settings to be configured.

The dialogues are virtually identical to those used to define a new SOP. To save repeating the same information here, please refer to Chapter 9 Managing SOPs, for more details.

Once all settings have been made select the Save as SOP... button, if required, to store the settings. Click the OK button to close the manual measurement settings dialogue and return to the measurement display.

Note.The manual measurement settings can be viewed and subsequently

saved by selecting Edit-Extract SOP.

Page 4.16 MAN 0317

The Measurement display

When an SOP or manual measurement is started the measurement display will appear showing the progress of the measurement.

The measurement display for all measurement types is generally the same and shows a number of dialogues representing the progress of the measurement sequence. The dialogues displayed is dependent upon the measurement type selected. The diagram below shows the display for a size measurement.

CHAPTER 4

The features of the measurement display are:

Button bar

The button bar provides the control for the measurement operation.

Settings

Opens the measurement settings dialogue. Extra comments and changes to the measurement parameters can be added prior to the measurement being started.

Start ($) and Stop (!) Starts and stops the measurement. If Stop is pressed while performing a measurement then the measurement must be started again from the beginning. Stop does not act like a pause.

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Zetasizer Nano Series

Help

Opens the Help file.

Closes the measurement display and returns to the record view. If close is pressed while a measurement is in progress the screen will close and all measured data will be lost; a warning box will appear asking “Are you sure you wish to abort the measurement”.

Status bar

The status bar shows instructions and the current operation in the measurement sequence, plus the temperature, measurement position and attenuator settings

Progress meter

The progress meter shows how far the measurement has progressed plus the number of measurements performed and the measurement runs completed.

Tab dialogues

The Tab dialogues alter depending upon the measurement type selected. The dialogues available are:

Size measurements

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

Count rate (Kcps)

Time

ILL 6756

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If dust is present then sharp spikes will be observed. Measurement runs with dust present will be removed from the final measurement calculation by a dust filtration algorithm.

A wildly fluctuating count rate may indicate that thermal gradients are present in the sample, and further time is required for temperature equilibration.

A steadily increasing count rate will indicate an aggregating sample, while a decreasing count rate will indicate a sedimenting sample.

Dust present

Count rate (Kcps)

Time

Thermal gradients

Count rate (Kcps)

Time

Aggregating sample Sedimenting sample

Count rate (Kcps)

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Log sheet

Displays the progress of the measurement.

Correlation function

The correlation function helps the experienced user to interpret any problems with the sample.

Time

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Large size

sample

Tim

= variable results

g(2)-1

Small size

sample

Tim

Contaminated

sample

Tim

g(2)-1

Noisy data

g(2)-1

Tim

Result

The result view will be updated after every run of the measurement. The result shown will be the sum of the acceptable data collected. When a result is available to view the result tab will turn blue.

Molecular weight measurements

Count rate

Please refer to size description above.

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Correlation function Please refer to size description above.

Result

The result view will be updated after each of the individual concentration measurements. The result shown will be an evolving value from the data collected so far.

Debye

Displays the current result as a Debye plot. The Debye plot displayed will show an evolving plot generated from the data collected so far.

Log sheet

Displays the progress of the measurement.

Zeta measurements

Count rate

Displays the number of photons detected in kilo-counts per second.

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Zeta potential

Displays the zeta potential result. The view will be updated after every individual measurement run, with the result being the sum of the completed measurement runs .

Log sheet

Displays the progress of the measurement.

Trend and protein melting point measurements

The trend and melting point dialogues are generally the same as those used with either a size or zeta measurement SOP is chosen. The difference is the inclusion of a Trend tab - this will show an evolving plot as the measurement progresses.

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.

Monitoring the status bar or clicking on the Log sheet tab will give more detail about what is happening during this procedure. The progress meter indicates how far the system is through the optimisation stages.

Once these stages have been completed, the measurement proper will start; again the actual measurement sequence will depend upon the measurement being performed.

Size measurements

The cell is inserted, Start is pressed and data collection begins. The progress meter indicates the measurement progress, while Measurement and Run show the number of runs completed and measurements performed.

The measurement is divided into a number of ‘runs’, This is done to allow data filtering. At the end of data collection the data quality of each ‘run’ is assessed; the runs that contain the poorest data are rejected while the remaining runs are analysed and used in the final measurement calculation.

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As soon as a run is completed the result tab changes to blue to indicate a preliminary size result is available to view (by clicking on the tab). As more runs are made and assessed the quality of the result will improve.

Molecular weight measurements

The molecular weight measurement sequence requires a series of intensity measurements to be made, first of a standard to establish the reference scattering intensity, and then of each of a number of prepared sample concentrations. At each part of the sequence the user will be prompted for the insertion of the next concentration. As this requires more interaction than for size and zeta potential measurements, the sequence has been described below

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 standard used.

Once the standard has been measured a dialogue box will appear to prompt insertion of the first sample concentration (i.e. the pure solvent). Insert the first sample concentration and press start.

The software displays another dialogue where the sample concentration can be entered. Type in the concentration and press enter. The measurement continues.

On completion of the first sample measurement, a dialogue is displayed answer Yes to “Repeat measurement of concentration 1?” or No to continue with the second concentration.

Continue as above until all sample concentrations have been measured.

On completion of the last concentration the final result will be calculated.

The progress meter indicates the measurement progress during each stage.

Zeta potential measurements

The cell is inserted and Start is pressed. The cell is first checked to identify the cell type fitted, and that it agrees with that selected in the SOP. Once identified the measurement sequence continues automatically. The status bar will indicate the instrument is now “Performing the measurement”.

Each complete measurement is divided into a number of measurement runs. All the individual measurement runs are accumulated together and then summed to give a final Zeta result.

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As soon as one measurement run is completed the zeta result tab changes to blue to indicate a preliminary zeta result is available to view (by clicking on the tab). As more measurements runs are made, the zeta result will change as more runs are accumulated and averaged until the final result is achieved.

Note

During a measurement sequence it is possible to select and view the information on any of the displayed tabs.

The final result is displayed and saved to the current open file. This report pages can be viewed when the measurement display has been closed.

To shorten the measurement sequence, select the ‘Auto’ measurement duration in the SOP; the change in zeta potential will now be monitored as the measurement progresses. The default number of runs in ‘auto’ measurement duration is 30, but for a stable sample as few as 10 runs may be required. The measurement will then complete even though the displayed run total has not been achieved.

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.

Right-click on a measurement record and select Edit Result; the dialogue below will appear. The Edit result tabs will show choices similar to those within the SOP editor.

Note.Each edit result dialogue will be slightly different depending upon the

Alter the appropriate parameter and press OK. It is advisable to add the modified parameter to the records list so the altered records can be instantly identified.

measurement type originally performed. The picture below shows the size view.

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If the 21 CFR part 11 feature is installed, a Reason for change dialogue will appear so comments can be entered detailing what changes have been made.

The reasons for change, can be displayed in the Records view tab by selecting the Measurement-Audit information-Reason in the Workspace settings dialogue ( Select Configure-Workspaces-“.. workspace choice..” and then the Record view parameters tab).

The result will instantly be reanalysed and the result added to the Record view.

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Records and Reports -

Viewing the results

CHAPTER 5

Introduction

Once a measurement has finished the results will need to be reviewed. This chapter details the displaying of the final measurement result.

The final result is displayed in the measurement file window as either a measurement record or report.

Displaying the results

The results are displayed in two ways. The records view that shows a list of the measurement records in a measurement file, and the reports tabs which show all measurement details of a selected measurement record.

CHAPTER 5

Note

The Records view parameters and Report tabs that are displayed are dependent upon the Workspace settings selected- refer to chapter 10.

Records view

Once the measurement is complete, a new measurement record will be shown in the Records view of the measurement file window. The records will be sequentially numbered. Records are automatically assigned a record number on completion of the measurement.

The parameters shown will be those selected using the Configure-Workspaces dialogues, the record view shown above depicts the default summary workspace (see chapter 10)

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Report tabs - a typical view

Selecting a measurement record and then selecting any of the report tabs will display the measurement information for that record.

This includes the pre-measurement settings such as sample name, the SOP and the SOP parameters used; the system settings for the measurement to be performed and the measurement results. A graph or table is also included at the bottom of the report.

Note

In addition to the information described in the body of the report, the footer of the printed report will show the software version and serial number of the 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.

Note

For each report two views are created. One view shows the printed version, the other view shows the computer screen version. This is done to accommodate for the different aspect ratios of the printed page and the computer screen.

To show multiple results on one report, hold down Shift or Ctrl and select the required records and then click the required report tab.

Size measurements - standard report

The standard report for Size measurements is Intensity PSD (M). (PSD stands for Particle Size Distribution).

The report is divided into four areas; these are described below.

Sample details

This section gives details of parameters relating to the sample. This includes the measurement name, record number, time of measurement, sample/dispersant refractive indices, viscosity, etc. The information shown is generally that which was entered by the user into the SOP measurement dialogues.

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System details

This section gives details on instrument settings for this particular measurement. Specifically, these are:

Temperature

Measured temperature at the start of the measurement.

Count rate

Average count rate for the measurement.

Measurement duration

This is the measurement time in seconds.

Cell type

This displays the cell type selected.

Attenuation index

The laser power is automatically attenuated so that the count rate from the sample, especially high scattering samples, is within acceptable limits. An attenuation index of 11 denotes no attenuation (full laser power), while 0 denotes full attenuation (total laser block). The attenuation range is shown in the following table; the transmission value is the percentage of laser light that enters the sample cuvette.

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onto

Zetasizer Nano Series

Attenuator Index

1 0.0003

2 0.003

3 0.01

4 0.03

5 0.1

6 0.3

910

10 30

11 100

Transmission (% Nominal)

Measurement position

The measurement position within the system is automatically moved to allow a large range of sample concentrations to be measured. The default measurement position is 4.65mm from the cell wall when a 12mm square cuvette is used. Numbers lower than this indicate that the measurement position is closer to the cuvette wall.

4.65mm 1mm

0mm

finstrument

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Note.The attenuator index and measurement position are automatically

adjusted by the software, though for more experienced users there is an advanced button that allows both to be set manually.

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.

The results section gives three pieces of information:

Z-average size (also known as the “cumulants mean”) In dynamic light scattering this is the most important and stable number produced by the technique. This is the size to use if a number is required for quality control purposes.

It will only be comparable with other techniques if the sample is monomodal (i.e. only one peak), spherical and monodisperse (i.e. no width to the distribution), and the sample is prepared in the correct dispersant.

In any other case, the Z-average size can only be used to compare results with samples measured in the same dispersant, by the same technique, i.e. by Dynamic Light Scattering (DLS).

The cumulants analysis only gives two values, a mean value for the size, and a width parameter known as the Polydispersity,orthePolydispersity Index (PDI). It is important to note that this mean size (often given the symbol Z or z-average) is an intensity mean. It is not a mass or number mean because it is calculated from the signal intensity.

The cumulants analysis is actually the fit of a polynomial to the log of the G1 correlation function.

Ln[G1] = a + bt + ct

+dt3+et4+ ...........

The value of b is known as the second order cumulant, or the z-average diffusion coefficient. This is converted to a size using the dispersant viscosity and some instrumental constants.

Only the first three terms a,b,c are used in the standard analysis to avoid over-resolving the data; however this does mean that the Z-average size is likely to be interpreted incorrectly if the distribution is very broad (i.e. has a high polydispersity).

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Polydispersity index

The coefficient of the squared term, c, when scaled as 2c/b

is known as the

polydispersity, or polydispersity index (PDI).

The calculations for these parameters are defined in the ISO standard document 13321:1996 E.

Peak means

Displays the size and percentage by either intensity, volume or number for up to three peaks within the result.

In summary, the cumulants analysis gives a good description of the size that is comparable with other methods of analysis for spherical, reasonably narrow monomodal samples, i.e. with polydispersity below a value of 0.1. For samples with a slightly increased width, the Z-average size and polydispersity will give values that can be used for comparative purposes. For broader distributions, where the polydispersity is over 0.5, it is unwise to rely on the Z-average mean, and a distribution analysis should be used to determine the peak positions.

Graph

The results are also shown in graphical form.

The format of the graph can be altered by moving the cursor over the graph and right clicking the mouse. The Graph control properties dialogue will appear. This dialogue allows the following attributes to be altered:

Display

The Display tab allows a choice of graph type and how it is to be displayed. i.e. Either as a histogram or curve, or particular statistics shown.

The Options tab allows the graph key position to be chosen. A graph tips option can also be set, this allows the setting of pop up tips (flags showing data points on the graph) on the report itself.

Axis settings

Allows both the X and Y axis settings to be defined. Whether logarithmic or linear axis are required and the axis scales - user defined or auto-scaling.

Graticule, or grid lines can also be shown on the graphs..

Font

Allows the font style to be altered. The setting will apply to all annotations used on the graph.

Upper/Lower limits Further tabs allow the setting of Upper and Lower warning or action limits so the value shown can be checked to be within desirable limits.

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Note.Though it is possible to change the appearance of the graph on the

screen, when the report is printed out it will print the original default view. To print out a different graph a new report will have to be created using report designer.

The graph below has been configured using the dialogue to display a size distribution by Intensity graph as a histogram, with logarithmic X-axis and linear Y-axis settings. Graticule, or grid, lines have been included and upper action and warning limits have been set.

It is possible to zoom into a graph report. Simply hold down the left mouse button, and move the mouse to draw a “Marquee” (from top left to bottom right) around the area to be enlarged. To zoom back out, simply click the left mouse button on the graph.

Other Size reports

Other Malvern views available for size measurements are:

Intensity Statistics (M)

Volume psd (M)

Volume Statistics (M)

Number psd (M)

Number Statistics (M)

Size diagnostics report (M)

This report shows six graphs: The size distribution by intensity and volume, size and cumulants residuals by time, and the data and cumulants fit. These graphs are helpful for more experienced users to determine the quality of their measurement data.

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Molecular weight measurements - standard report

The standard report for molecular weight measurements is Molecular weight report (M).

The report is divided into four areas; these are described below.

Sample

The Sample section gives details of parameters relating to the sample.

Please see the size description for details.

System details

The System section gives details on settings configured during the measurement process.

Please see the size description for details about Temperature, Cell type and Run duration.

Settings specific for molecular weight are:

Dn/Dc

This is the differential refractive index increment; the change in refractive index as a function of the change in concentration.

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Shape model

Shows the shape correction model used for the measurement. With knowledge of the sample configuration it is possible to improve the result of the measurement by adding the value that most closely corresponds to the probable sample shape; i.e. sphere, coil, cylinder or no shape correction.

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 in Daltons.

Shape correction

Displays the shape correction model used for the calculation as mentioned above.

Second Virial Coefficient

A property describing the interaction strength between the molecule and the solvent.

Fit error

This is an indication of the quality of the measurement. The lower the fit error the better the measurement.

Graph

The results are also shown in graphical form.

Please see the size description above for details on altering the graph.

Zeta potential measurements - standard report

The standard report for zeta potential measurements is Zeta potential (M).

The report is divided into four areas; these are described below.

Sample

The Sample section gives details of the sample parameters. This includes the sample name, record number, measurement date and time, dispersant name, The SOP used and the measurement file name. The information shown is generally that which was inputted into the SOP measurement dialogues.

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System

The system section gives details on settings configured during the measurement process.

Please see the size description for details about Temperature, Cell type, Run duration and Attenuator.

Settings specific for zeta measurements are:

Mean Count rate

Average count rate for the measurement.

F(ka) value

Displays the Henry function or ‘approximation’ used during the measurement. A value of 1.5 means the Smoluchowski approximation was used, while a value of 1 means the Huckel approximation was used.

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Results

The results section gives four pieces of information:

Zeta potential

Displays the complete zeta potential result of the measurement in mV.

Std Dev. (Standard deviation).

The standard deviation displays 1 standard deviation of the zeta distribution around the mean result, in millivolts (mV).

Conductivity. The ability of a sample to conduct electrical current. Higher salt concentrations generally allow higher currents to pass and so have higher conductivities. Conductivity can be temperature dependent.

Peak means. Displays the mean zeta potential for up to three peaks within the result.

Graph

The results are also shown in graphical form.

Please see the size description above for details on altering the graph.

Other Zeta potential reports

Other Malvern views available for zeta potential measurements are:

Electrophoretic mobility (M)

This report is effectively the same as the zeta potential report described, except the zeta potential result and graph are replaced by the electrophoretic mobility.

Trend and protein melting point measurements

These measurements can be viewed using the report Melting point (M). The report gives the same information as seen in a standard size or zeta report, plus the following:

Trend Temperature Start (°C)

The temperature defining the beginning of the measurement.

Trend Temperature End (°C)

The temperature that the measurement will end at.

Melting point (°C)

The temperature at which the melting point is acheived.

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Sample Preparation

CHAPTER 6

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. For each measurement type follow the guidelines described.

Preparing the sample - Size

Consideration must be given to the physical properties of the sample such as its particle size and sample concentration. This section outlines the basic considerations for sample preparation.

Sample concentration

Each type of sample material has its own ideal range of sample concentration for optimal measurements.

If the sample concentration is too low, there may not be enough light scattered to make a measurement. This is unlikely to occur with the Zetasizer except in extreme circumstances.

If the sample is too concentrated, then light scattered by one particle will itself be scattered by another (this is known as multiple scattering).

The upper limit of the concentration is also governed by the point at which the concentration no longer allows the sample to freely diffuse, due to par ticle interactions.

CHAPTER 6

An important factor in determining the maximum concentration the sample can be measured at, is the size of the particles.

The table below can be used as an approximate guide to determine the maximum and minimum concentrations for different sizes of particles. The figures given are approximate values for samples with a density near to 1g/cm particles have a reasonable difference in refractive index to that of the dispersant, e.g. a refractive index of 1.38 against water which has a refractive index of 1.33.

, and where the

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Particle size Min. Concentration

(Recommended)

< 10nm 0.5g/l Only limited by the sample material

10nm to 100nm 0.1mg/l 5% mass (assuming a density of 1g/cm

-3

100nm to 1µm 0.01g/l (10

> 1µm 0.1g/l (10

% mass) 1% mass (assuming a density of 1g/cm3)

-2

% mass) 1% mass (assuming a density of 1g/cm3)

Max. Concentration

(Recommended)

interaction, aggregation, gelation etc.

Whenever possible, the sample concentration should be selected such that the sample develops a slightly milky appearance - or in more technical terms, gets slightly turbid.

If such a concentration cannot be selected easily (for example, the particle size of the sample may be so small that even concentrated dispersions show no turbidity), various concentrations of the sample should be measured in order to detect and then avoid concentration dependent effects (i.e. particle interactions etc). A concentration should be chosen such that the result is independent of the concentration chosen. However, these effects do not normally appear at concentrations below 0.1% by volume.

)

Be aware that particle interactions may occur at sample concentrations larger than 1% by volume - particle interactions will influence the results.

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 dispersant. As a guide, the scattering from water should give a count rate in excess of 10kcps, and toluene in excess of 100kcps.

Maximum concentration

For samples with small particle sizes, a maximum concentration does not really exist (in terms of performing Dynamic Light Scattering (DLS) measurements). However, in practice, the properties of the sample itself will set the maximum value. For example, the sample may have the following properties:

Gelation. A gel is unsuitable for measurement using the Zetasizer (this is true for all instruments based on Dynamic Light Scattering).

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Particle interactions. If there are interactions between the particles then the diffusion constant of the particles usually changes, leading to incorrect results. A concentration should be chosen to avoid particle interactions.

Considerations for large particles

Minimum concentration

Even for larger particles, the minimum concentration is effectively still a function of the amount of scattered light, though the additional effect of “number-fluctuation” must be taken into account.

As an example, if a sample of large particles (say 500nm) were to be measured at low concentration (say 0.001 g/l (10

-4

%)), the amount of scattered light generated would be more than sufficient to perform a measurement. However, the number of particles in the scattering volume is so small (fewer than 10) that severe fluctuations of the momentary number of particles in the scattering volume will occur. These fluctuations are not the type assumed by the calculation method used, or will generally be misinterpreted as larger particles within the sample.

Such fluctuations must be avoided and this determines the lower limit for the required concentration and for a lower limit in the number of particles. At least 500 particles should be present, however, a minimum of 1000 particles is recommended. See the figure below for an estimate plot of the number of particles per scattering volume for different concentrations assuming a density of 1 g/cm³.

1.00E+13

1.00E+12

1.00E+11

1.00E+10

1.00E+09

1.00E+08

1.00E+07

Particles

1000000

100000

Number o

10000

1000

100

0.1

1nm 10nm 100nm 1µm 10µm

1 g/l

0.1 g/l

0.01 g/l

0.001 g/l

100 particles

Particle Diameter

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Maximum concentration

The upper limit for the sample concentration for larger particles is determined by their tendency to cause multiple-scattering. Although the Zetasizer is not very sensitive to multiple-scattering, with increased concentration, the effect of the multiple-scattering becomes more and more dominant, up to a point where so much multiple-scattering is generated that the results of the measurement will be affected. Of course, such a high concentration should not be used for accurate measurements, and rough estimates are given for maximum concentrations for the different size classes in the table above.

As a general rule, measure at the highest concentration possible before multiple scattering and particle interactions affect the result. Dust contamination in the sample can be presumed equal for high and low concentrations, and thus, the amount of scattered light from the sample increases in relation to the scattering from the dust contamination as the concentration increases.

Filtration

All liquids used to dilute the sample (dispersants and solvents) should be filtered before use to avoid contaminating the sample. The size of the filter will be determined by the estimated size of the sample. If the sample is 10nm, then 50nm dust will be an important contaminant in the dispersant. Aqueous dispersants can be filtered down to 0.2µm, while non-polar dispersants can be filtered down to 10 or 20nm.

Samples are not filtered if at all possible. Filters can remove sample by absorption as well as physical filtration. Only filter the sample if aware of larger sized particles, such as agglomerates, that need to be removed as they are not of interest, or cause result variations.

Using ultrasonics

Ultrasonication can be used to remove air bubbles or to breakup agglomerates however, this must be applied carefully in order to avoid damaging the primary particles in the sample. Limits for the use of ultrasonication in terms of intensity and application time are strongly sample dependent. Minerals such as titanium dioxide are ideal candidates for dispersion by high powered probes, however, the particle size of some materials, such as carbon black, may depend on the power and length of time ultrasonication is applied. Some materials can even be forced to aggregate using ultrasound.

Emulsions and liposomes should not be ultrasonicated.

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Preparing the sample

- Molecular weight

Preparation of a molecular weight sample is similar to that followed for a size sample, though there are other aspects to consider.

The technique is very sensitive to dirt or dust in the sample and therefore great care is required in sample preparation. All solvents must be filtered to 0.02µm or better several times, and the prepared solutions allowed to stand for a period dependent upon the sample, this may be over 24 hours, to several days to ensure adequate solvation. All glassware and apparatus must be rigorously clean and free of scratches. Preparation of samples and storage of apparatus in a laminar flow cabinet to ensure minimisation of dust contamination is strongly recommended. Failure to carry out these routine procedures are certain to end in a poor or wrong result.

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

-1

). The polymer must be fully soluble and dust must be excluded.

CHAPTER 6

Solvent

Increasingsample concentration

1 2 3 4

The solvent scattering is measured followed by the various concentrations of sample. From these measurements a Debye plot can be generated.

Intercept point

Concentration

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Preparing the sample - Zeta potential

Zetasizer Nano Series

This is a plot of the variation in average intensity versus the concentration. The intercept of the extrapolation to zero concentration is calculated.

Minimum concentration

The minimum concentration of sample that should be used is defined by the excess scattering over the solvent which should be a minimum of 30%, e.g. If the solvent is toluene with a count of 150 Kcps then the lowest sample concentration should be greater than 150 x 1.3 kcps (195Kcps). Careful sample preparation procedures can make it possible to measure a sample with only 10% excess scattering but this is not ideal.

Maximum concentration

The maximum concentration will be sample dependent and will be determined by the onset of particle interactions. Typically it is best to keep the maximum concentration to below 0.1w/v%

The optical arrangement for zeta potential measurements in the Zetasizer Nano series means that the concentration requirements are not as strict as those used for size and molecular weight measurements.

An auto attenuator fitted inside the instrument ensures that the sample count rate is suitable for the requirements of the detector. However if the sample is too concentrated a low sample count rate error will be displayed. For example, for a 300nm oil in water emulsion this will be the case for concentrations higher than

0.5w/v%.

Too low a concentration will be indicated by the system setting an attenuator index position of 11, and the generated results being variable.

In extreme cases, or where there is a bubble in the cell, a ‘low sample concentration’ error may be displayed.

Many samples will require dilution and this procedure is absolutely critical in determining the final value measured. For meaningful measurements the dilution medium is crucially important. A measurement result given with no reference to the medium in which the material is dispersed is meaningless. The zeta potential is as dependent on the composition of the disperse phase as it is on the nature of the particle surface.

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Dilution Medium

The continuous phase of most samples can arbitrarily be put into one of two categories:

Polar dispersants are defined as those with a dielectric constant greater than 20 e.g. ethanol and water.

Non-polar or low polarity dispersants are defined as those with a dielectric constant less than 20, e.g. hydrocarbons, higher alcohols.

Aqueous/Polar Systems

The aim of sample preparation is to preserve the existing state of the surface during the process of dilution. There is only one way to ensure this is the case. This is by filtering or centrifuging some clear liquid form the original sample and using this to dilute the original concentrated sample. In this way the equilibrium between surface and liquid is perfectly maintained.

If extraction of a supernatant is not possible, then just letting a sample naturally sediment and using the fine particles left in the supernatant is a good method. Zeta potential is not a size-dependent parameter using the approximation of the Smoluchowski theory.

Another method is to imitate the original medium as closely as possible. This should be done with regard to:

pH.

Total ionic concentration of the system.

Concentration of any surfactants or polymers present.

Non-Polar Systems

Measuring samples in insulating media such as hexane, isoparaffin, etc. is far from easy. It requires the use of the universal dip cell. This is required because of its chemical compatibility and the close spacing of the electrodes which allows the generation of high field strengths without using excessively high voltages.

Sample preparation for such systems will follow the same general rules as for polar systems. As there will be generally fewer ions in a non polar dispersant to suppress the zeta potential, the actual values measured can seem very high, as much as 200 or 250mV. In such non-polar systems, equilibration of the sample after dilution is the time dependent step, equilibration can take in excess of 24 hours.

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Zetasizer Nano Series

Page 6.8 MAN 0317

Malvern ZEN3600, ZEN2600, ZEN1500, ZEN2500, ZEN3500 User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual