Malvern Zetasizer uV User Manual

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Malvern

Zetasizer μV

User Manual

MAN0428 Issue 1.0 December 2008

Malvern 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

Zetasizer is a registered trademark in the UK and /or other countries, and is owned by Malvern Instruments Ltd. Malvern and the green “hills” logo are registered trademarks in the UK and/or other countries, and is owned by Malvern Instruments Ltd.

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

Windows 2000 and XP are registered trademarks of the Microsoft Corporation. Zetasizer, NIBS and M3-PALS are registered trademarks of Malvern Instruments. Tygon is a registered trademark of Cole Palmer Instruments Company Limited.

Printed in England

Addendum to manual

Addendum number ADD0076-1.0

Date 04/02/2009

Manual number MAN0428-1.0

Manual name Zetasizer μV User Manual

This addendum presents new information for taking measurements at ambient temperatures while using the Flow-mode facility on the Zetasizer

The information below should be inserted as a third paragraph in the Sample -

Temperature section on page 9.16, and as a fifth paragraph in the Flow-mode SOPs section on page 9.33.

Note

To improve the stability of temperature control when using the system as a Gel Permeation Chromatography (GPC) detector, the measurement temperature should be set to be 5°C or greater, above or below the ambient temperature.

μV instrument.

Addendum 0076-1.0 Page 1

Page 2 Addendum 0076-1.0

Addendum to manual

Addendum number: ADD0081-1.0

Date: 26/05/2010

Manual number: MAN0428-1.0

Manual name: Zetasizer V User Manual

This Addendum presents new information for operating the Zetasizer V instrument. The content should be read in-place of, or in conjunction with, the sections indicated in the respective manual. The information detailed will be incorporated into the next manual release for the instrument.

The Zetasizer μV measurement range - Read in place of relevant section on page 2-1 of the Zetasizer μV User Manual.

The Zetasizer μV measurement range

The Zetasizer V measurement specifications are described below:

 Size range maximum (radius): 0.15nm to 1micron

 Molecular weight range: 342Da to 2x10

(est from hydrodynamic diameter)

Molecular weight range: 980Da to 2x10 (calculated from Debye plot)

For full measurement specifications refer to Appendix 1.

Addendum 0081-1.0 Page 1

Choosing the correct cell - Read in place of relevant section on page 4-3 of the Zetasizer μV User Manual.

Choosing the correct cell

Caution!

Due to the risk of melting, polystyrene cuvettes must not be used for measurements above 70°C.

Malvern offers a range of cells for performing measurements with the Zetasizer The 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.

Size measurements - Read in place of relevant section on page 4-4 of the Zetasizer μV User Manual.

V.

Size measurements

Disposable polystyrene (DTS0012)

Typical solvent Water, Water/ethanol

Optical quality Good to very good

Minimum Sample volume

Advantages Low cost

Disadvantages Not resistant to organic

Material Polystyrene

1ml

Single use disposable (no cleaning)

solvents

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

Page 2 Addendum 0081-1.0

Specification - Read in place of relevant section on page A-1 of the Zetasizer μV User Manual.

Specification

Parameter Specification

Size range maximum (radius) * 0.15nm to 1micron

Molecular weight range ** (est from hydrodynamic diameter)

Molecular weight range ** (calculated using Debye plot)

Minimum sample volume 2µL

Sensitivity at minimum volume 0.1mg/ml of 15kDa protein

High concentration Yes - optically clear samples

SEC detector mode 8µl flow cell

Laser 60mW, 830nm

Temperature range 2°C to 90°C ±0.1 °C

Temperature accuracy 0.1°C at 25°C, 0.2°C at 2°C, 0.5°C at 90°C

Compatible cell temperatures 2°C to 90°C -- Glass and quartz cells

Weight 14kg

Dimensions W:D:H, 350mm x 410mm x 170mm

Power 90V-260V AC, 50/60Hz, 100W

Dry air 100kPa (1bar), 4mm o.d. tubing

Computer interface USB

Measurement technique Dynamic light scattering (90 degrees)

Detector Avalanche photodiode, Q.E. >50% at

Product laser class Class 1 compliant,

Condensation control Purge facility using dry air

Ambient operating conditions

- Humidity

342Da to 2x10

980Da to 2x10

2°C to 70°C -- Plastic disposable cells

830nm

IEC 60825-1(1993)+A1(1997)+A2(2001)

+10 to +35 °C (+50 to 95 °F) 10 to 90% (non-condensing)

* Peak mode range (radius), 0.3nm - 0.5 microns, sample dependent ** Sample and dependent

Addendum 0081-1.0 Page 3

Page 4 Addendum 0081-1.0

Table of contents

Part 1 - Operator’s Guide

Introduction to this manual

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

How to use this manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

Access to the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Assumed information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-3

Where to get help. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-4

What is the Zetasizer μV?

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

What does the Zetasizer μV do? . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

What are Particle Size and Molecular weight? . . . . . . . . . . . . . . . . 2-2

How does the Zetasizer μV work?

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

How is a Zetasizer measurement performed? . . . . . . . . . . . . . . . . . 3-2

What does the Zetasizer consist of? - Identifying the hardware . . . . 3-5

Navigating the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-11

A Tutorial - Making a measurement

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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-7

Inserting the cell. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-9

Making an SOP measurement. . . . . . . . . . . . . . . . . . . . . . . . . . . 4-11

Making a manual measurement . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Zetasizer μV Page i

Table of Contents Zetasizer μV

The Measurement display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-13

Records and Reports - Viewing the Results

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

Displaying the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-1

Sample Preparation

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

Preparing the sample - Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

Preparing the sample - Molecular weight . . . . . . . . . . . . . . . . . . . . 6-2

Count rate meter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-4

Part 2 - Supervisor’s Guide

Security

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-1

Initial start-up - set up the administrator. . . . . . . . . . . . . . . . . . . . . 7-2

Enabling security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

User groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-4

Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-5

Measurement file window - Workspaces

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1

Measurement file window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2

Using SOPs

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1

The SOP Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-3

Creating an SOP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-8

Measurement type selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-9

Size SOPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-10

Molecular weight SOPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-25

Flow-mode SOPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-33

Trend SOPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-35

Page ii MAN 0428

Zetasizer μVTable of Contents

Extracting an SOP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-37

Modifying an SOP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-38

Distributing an SOP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-38

Advanced features

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-1

Solvent Builder. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-2

SOP Player . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-4

Result averaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-9

Editing the result . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-10

Exporting results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-12

Flow-mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-16

Options dialogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-21

Expert Advice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10-25

Creating Custom Reports

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-1

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-2

Opening an existing report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-3

Creating a new report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-3

Adding elements to the report . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-4

Setting up the report elements . . . . . . . . . . . . . . . . . . . . . . . . . . 11-5

Selecting elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-13

Aligning and sizing elements . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-14

Saving a report. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-14

A finished report. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-15

Viewing the new report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-15

Other information shown on a report . . . . . . . . . . . . . . . . . . . . . 11-16

Protecting a report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11-16

Protein Utilities

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-1

Debye plot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-2

Molecular weight calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-7

Concentration utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-10

Zetasizer μV Page iii

Table of Contents Zetasizer μV

Scattering functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12-11

Size theory

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13-1

What is Dynamic Light Scattering? . . . . . . . . . . . . . . . . . . . . . . . 13-1

Operation of the Zetasizer μV - Size measurements . . . . . . . . . . . 13-6

Molecular Weight theory

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14-1

What is Static light scattering? . . . . . . . . . . . . . . . . . . . . . . . . . . 14-1

The Debye plot. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14-4

Part 3 - Appendices

Specification

Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

Chemical compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-2

Unpacking instructions

Installation

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

Installing the Zetasizer μV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2

Changing the computer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-2

Regulatory Statements

Disposal of Electrical & Electronic Equipment . . . . . . . . . . . . . . . . . D-1

FCC Notice (US only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

Canadian Regulatory Information. . . . . . . . . . . . . . . . . . . . . . . . . . D-3

VCCI acceptance (Japan only). . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3

Page iv MAN 0428

Part 1 Operator’s Guide

Introduction to this manual

Introduction

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

Note

The Zetasizer model, Serial number, software and firmware version can be found by left-clicking the μV icon in the right corner of the status bar.

The aim of this manual is to:

 Identify what the instrument is.

 Explain in simple terms how it works.

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

 Identify the user maintenance procedures.

How to use this manual

Read this manual together with the Essentials manual, which describes Site Requirements, Health and Safety and Maintenance information.

Warning!

The instrument and the samples to be measured may be hazardous if misused. Users must read the Health and Safety information in the Essen- tials manual before operating the system.

Zetasizer μV Page 1-1

Chapter 1 Introduction to this manual

We recommend reading both these manuals fully before starting the first measurement, though if more familiar with particle size analysers, jump straight to Chap- ter 4. This chapter gives practical details on making measurements.

This manual is divided into three sections as follows.

Part 1 – Operator’s guide

This guide and the Essentials manual contain all the information required for the operator to use the Zetasizer μV instrument.

Topics covered are: What is the Zetasizer μV instrument, what are the components of the Zetasizer μV, what it does, instructions on using the instrument and the software, basic measurement and maintenance procedures.

Part 2 – Supervisor’s guide

The Supervisor’s guide concentrates on the administration of the Zetasizer μV and covers some of the more advanced features. It also provides a greater insight into the measurement procedures and expands on the analysis theories.

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

It is recommended that the supervisor should also read Part 1 – Operator’s guide.

Part 3 - Appendices

This contains supplementary information not necessary for the general operation of the system.

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

Page 1-2 MAN 0428

Introduction to this manual Chapter 1

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!

Removal of the main covers by unauthorised personnel, even a supervisor, will invalidate the warranty of the instrument.

Supervisor

The supervisor is responsible for the management and safety of the instrument and its operation. The supervisor also trains the operators. They can perform all user maintenance routines identified in the Essentials manual.

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 the Essentials manual, except changing the fuse.

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

Warning!

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

Assumed information

Naming convention

The Zetasizer μV will either be referred to in full, or as “the instrument”.

Zetasizer μV Page 1-3

Chapter 1 Introduction to this manual

The combination of the Zetasizer μV instrument, the computer and Zetasizer software may be referred to as the “the system”.

Cells and Cuvettes

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

Solvents and dispersants

To reflect the intended protein analysis applications of the system, the term “solvent” is used throughout this manual to refer to any fluid used to disperse a sample’s particles for measurement. However, solvent can also be taken to mean “dispersant” where this is more applicable to the sample type.

Menu commands

Menu commands from the Zetasizer software are referred to in the form main menu-menu item. As an example, the command Tools-Options refers to select-

ing the Options item in the Tools menu. Menu commands are shown in bold text.

Where to get help

Manuals and Help files

The primary sources of help for the Zetasizer μV are the Essentials manual, this manual and the on-line help system within the software. This manual is designed to give an overview of the system as a whole, while the on-line help system is designed to give detailed information on the Zetasizer software.

Each dialogue within the Zetasizer software has a Help button that gives information specific to that dialogue.

Essentials Manual

This manual covers:

 Site requirements – all the physical requirements for positioning the system.

Information is given on; services requirements (air, water, number of power sockets, etc.), environmental requirements (temperature, humidity etc.) and physical requirements (space requirements etc.).

 Health and safety – this must be read by all users of the system. It details all

safety issues for the optical unit and all accessories.

Page 1-4 MAN 0428

Introduction to this manual Chapter 1

 Maintenance – for both the optical unit and accessories.

Help desk

All queries regarding the system should initially be directed to the local Malvern representative. Please quote the following information:

 Model and serial number of the instrument (located on the rear panel).

 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. This help line is primarily English speaking.

Remote support

Malvern Instruments offers a remote support service over the Internet. 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 must be available to make use of this facility.

Malvern Website - www.Malvern.com

The Malvern website offers a comprehensive range of particle characterisation resources for use by customers 24 hours a day, seven days a week.

Resources include software downloads, frequently asked questions, a knowledge base and application notes, plus information on other particle characterisation solutions that Malvern can provide.

Zetasizer μV Page 1-5

Chapter 1 Introduction to this manual

Page 1-6 MAN 0428

What is the Zetasizer μV?

Introduction

This chapter gives a brief overview of the Zetasizer μV instrument and simple explanations about the measurement technique.

What does the Zetasizer μV do?

The Zetasizer μV instrument provides the ability to measure two characteristics of particles or molecules in a liquid medium.

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

The Zetasizer μV features pre-aligned optics, plus the precise temperature control necessary for reproducible, repeatable and accurate measurements. In addition, the facility is included for measurements of other key parameters such as 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) alleviate the need for constant attention.

The Zetasizer μV measurement range

The Zetasizer μV measurement specifications are described below:

 Size range radius: 0.3nm to 0.5μm

 Molecular weight range: 380Da to > 20MDa

Zetasizer μV Page 2-1

Chapter 2 What is the Zetasizer μV?

10 100

Time

10 100

Time

What are Particle Size and Molecular weight?

This section describes the basics of Particle Size and Molecular weight are and why they are important. Greater detail on the instrument’s measurement techniques is given in the chapters that cover theory (Chapter 13 and Chapter 14).

What is Size?

The Particle Size measured in a DLS instrument is the diameter or radius of the sphere that diffuses at the same speed as the particle being measured.

The Zetasizer μV 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 or molecules in a liquid due to the bombard-

ment by the solvent 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 or molecule.

Small particles moving quickly

1000

Size nm

Large particles moving slowly

1000

Size nm

ill 6722

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

Page 2-2 MAN 0428

What is the Zetasizer μV? Chapter 2

1.0081.008

15.999

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

The above is a very simplistic explanation. A more detailed explanation can be found in Chapter 13.

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

O (water) we can work out the

molecular weight.

 In each molecule of water there are two atoms of hydrogen (H

) 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.

18.015

ill 6721

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

With the Zetasizer μV instrument, the molecular weight can now be determined by use of Static Light Scattering (SLS) measurement techniques.

This technique is explained in Chapter 14.

Note

Malvern uses Daltons as the unit of molecular weight. Results are displayed in kiloDaltons (kDa).

Zetasizer μV Page 2-3

Chapter 2 What is the Zetasizer μV?

Why do we want to know it?

We need to know the molecular weight, so we can determine how many grams there are in 1 mole of a substance. The mole is the chemistry standard term for ‘1 molecular weight’, e.g. one mole of water is 18.015g.

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Introduction

Previously we identified the instrument and described the various measurement processes that can be performed. This chapter introduces the hardware and software features that the instrument incorporates.

The initial section, “How is a Zetasizer measurement performed?”, will briefly describe the following:

 What is involved in making a measurement?

 What are the major components of the system?

 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 and molecular weight measurements will be described in later chapters.

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Chapter 3 How does the Zetasizer μV work?

How is a Zetasizer measurement performed?

The Zetasizer system comprises the Zetasizer instrument  and a computer with the Zetasizer software installed into the cell area on the top of the instrument

. A cell  is filled with the sample and loaded

.

ill 6744

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 tem-

plate 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 reduce the requirement for repetitive user input and the risk of introducing 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 (see below). The measurement is begun 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.

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How does the Zetasizer μV work? Chapter 3

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. After

choosing these, begin the measurement by simply pressing the Start button on the Measurement display (below).

Once the measurement is complete the results can be viewed; either in a Record

view

, by selecting one of the Malvern pre-set reports , or a user defined report

.

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Chapter 3 How does the Zetasizer μV work?

UPDATE

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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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How does the Zetasizer μV work? Chapter 3

What does the Zetasizer consist of?

- Identifying the hardware

The diagram below shows a typical system with its key modules, the Zetasizer instrument and a computer system with the Zetasizer software installed. It is preferable the computer is dedicated to just running the Zetasizer software.

ill 8306

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

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Chapter 3 How does the Zetasizer μV work?

The Zetasizer μV instrument

 Optical unit

Positioned on the cover is a label that identifies the instrument model.

 Rear Panel

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

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! Power input socket

ill 8308

Mains power input socket for the instrument.

" Computer connection

The USB cable from the computer is connected here.

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How does the Zetasizer μV work? Chapter 3

# Purge connection

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 likely in humid climates. If it is suspected that this may be a problem then the purge inlet port can be used to connect a dry-air supply - i.e. a supply with a dew point below that of the target temperature. This will remove any moisture in the air immediately surrounding the cell and prevent condensation.

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

 Compressed air to DIN 8573-1

 Oil = Class 1

 Water = Class 3

 Particulate = Class 3

 Pressure = 100 kPa g

For connection purposes, the purge input uses a push-fit connector.

An optional purge connection kit is available from Malvern instruments if this facility is to be used.

When performing a purge measurement the following rules should be taken into account:

 The flow rate will be preset at 1.5ltr/min at 1 bar.

 The instrument should be operated at a maximum of 30°C & 70% relative

humidity when the requested temperature is below the dew point.

 When operating at a humidity higher than 50%, the measurement should allow

a minimum delay of 4 minutes to allow the temperature to stabilise.

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.

$ Power switch

The on/off power switch for the instrument. When switched on, the blue power indicator

Zetasizer μV Page 3-7

 will illuminate.

Chapter 3 How does the Zetasizer μV work?

Warning!

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 μV model and its serial number. Please quote all numbers in any correspondence with Malvern Instruments.

 Power light

Illuminates blue when the system is powered on.

 Flow mode tubing outlet

A channel in the cell area lid provides the option to connect flow cell tubing. See Chapter 10 for more information on flow mode.

 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.

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.

Chemical compatibility

Apart from the measurement cells, the only part of the instrument that should come into contact with any sample is the cell area if spillage occurs. The cell area has been manufactured from materials that are considered to give the widest protection from chemical attack. However, it is important to check that any sample used is chemically compatible with the materials from which the cell area is manufactured.

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How does the Zetasizer μV work? Chapter 3

Refer to Appendix A for a list of the cell area components that may come into contact with any spilt sample.

Features of the cell area

Other important features of the cell area are described below.

ill8310

! Cell area lid

Press down on top of the cell area lid (where it is marked “Push”) to unlock the catch, and then release. The lid will rise to an upright, open position allowing access to the cell holder.

 Laser safety interlock. This is activated on opening the cell area lid and pre-

vents any laser light from entering into the cell area.

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

Note

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

" Cell basin

The cell basin is made of an insulating material which provides protection from the heated cell holder.

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

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Chapter 3 How does the Zetasizer μV work?

# Flow tube channel

Channel provides access for a tube when an optional flow cell is connected.

$ Flow tube notch

Notch provides access for a tube when an optional flow cell is connected.

% Cell lid catch

Mechanism to keep the cell closed during a measurement.

Note

It is recommended that when using glass or quartz cuvettes, to first insert a disposable polystyrene cuvette into the cell holder. This is done to ensure the cell clamp moves freely, and that no spilt sample has caused the mechanism to stick.

Note

The Zetasizer μV model, serial number, software and firmware version can be found by double-clicking the μV icon in the right corner of the status bar.

 Cells and Cuvettes

A range of cells and cuvettes are available to use with the Zetasizer instrument. Full details are given in the Chapter 4, but briefly the following cells can be used.

Cell Application

Disposable “polystyrene” cuvettes – Standard and Small volume

Quartz glass cuvettes – Square, Standard, Low and ultralow volume, flow

Size

Size and Molecular weight

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How does the Zetasizer μV work? Chapter 3

Navigating the software

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

There are two software modules:

 The main Zetasizer application which is described below

 A secondary module known as Report Designer which enables custom reports

to be created to display the results. The features of Report Designer are detailed in Chapter 11.

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

The Zetasizer software application

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

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Chapter 3 How does the Zetasizer μV work?

 Menu bar

The menu bar contains the main menu headings for all software functions.

Items which end with a row of dots (...) will cause dialogue boxes to appear. Similarly any items which end with an arrow (

 ) will cause a second menu to

appear.

Items that are shown in grey 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 can be used to either create a New Measurement or SOP file, or Open... an existing Measurement or SOP 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.

 An SOP file contains all the measurement settings required for performing

repetitive and consistent measurements.

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.

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How does the Zetasizer μV work? Chapter 3

As a shortcut, a list of the most recently used measurement files are shown at the bottom of the menu for immediate opening.

Exit will close down the software.

Edit Menu

This allows movement and manipulation of records in the Measurement file window(s). 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 solvent and particle properties. Comments on the editing reason can be 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 editor 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.

Create Average Result, enables a number of records to be selected, then recalculated to show the average result. The averaged result is added to the bottom of the record view.

Note

A combination of the Edit and View menus will appear when the right mouse button is pressed anywhere in the measurement file window.

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Chapter 3 How does the Zetasizer μV work?

View Menu

The View menu allows the selection of which reports will be shown in the measurement file window and those Toolbars to be displayed.

To see the Standard deviation, the Relative standard deviation, the minimum, the maximum or the mean of a selected number of records, a Statistics bar can be displayed.

Additionally View type will display the Melting point in either a Sorted or Outlined format.

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.

All currently available reports are shown at the base of the menu. The icon

indicates the report that is currently displayed.

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

Expert Advice enables a quality check to be performed on three or more completed measurement records. This will show how good the measurements are and whether they display any unwanted attributes, such as aggregation. The Expert advice is also available directly from the records view, and during a measurement sequence in the measurement display. Refer to the Expert Advice section for a description.

Plate Navigator provides a graphical view of measurements made using a Zetasizer APS instrument. Greyed out for results made on the Zetasizer μV.

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How does the Zetasizer μV work? Chapter 3

Note

A combination of the Edit and View menus will appear when the right mouse button is clicked anywhere in the measurement file window.

Measure Menu

Select this menu when ready to perform a measurement.

There is a choice of using an existing measurement SOP (Measure-Start SOP...) or manually setting up the measurement and sample details (Measure-Manual).

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

The most recent SOPs used are displayed at the base of the menu.

SOP Player opens the SOP Player program; this enables a sequence of SOPs and other actions to be performed sequentially. See the SOP Player section for a description.

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Chapter 3 How does the Zetasizer μV work?

Too ls Me n u

The operation of Report Designer is explained in Chapter 11.

The Utilities-Protein option is described in Chapter 12. With this tool, estimates of the Molecular weight and particle shape can be generated.

The Count Rate Meter displays the number if photons detected per second. This is useful for determining both the sample quality and concentration. Refer to the Count rate meter section at the end of Chapter 6.

The Macros submenu will show additional available programs. These are programs written by Malvern to enable the system to operate in a mode required for a specific application. These can be installed and selected using the Options feature.

The Instrument Sub-menu allows access to the manual controls of the instrument or each connected accessory.

 Serial port... allows the USB communications port to which the instrument is

connected to be chosen. This menu item is greyed out - available only on the Zetasizer Nano series.

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

The Settings sub-menu provides access to the Configure Workspace, Data export templates and Line styles options.

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How does the Zetasizer μV work? Chapter 3

 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, workspaces to be imported or exported, and previously created

workspaces to be displayed.

 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.

Options opens a dialogue that allows the following options to be selected:

 Startup - Allows the user to specify how the software behaves on startup.

Select the Auto login... option to circumvent the standard Zetasizer log in window (and automatically log in as the current Windows user).

 Folders - Indicates and defines where the measurement data, SOPs and

Export data are saved.

 Macros - Enables the selection and installation of additional programs.

 Feature Keys - This page allows extra features of the Zetasizer software to be

enabled using a licence key.

 Measurement - This allows the look of the measurement display to be

altered.

 Instrument type - This allows the selection of instrument type (Nano, μV or

APS). The system remembers this setting and uses the same mode the next time the software is started.

Security Menu

To prevent unauthorised changes, the Malvern software can be configured to limit each user’s access to various functions - e.g. modifying an SOP. Users are assigned operating permissions that allow, or restrict, access; refer to Chapter 7 for details.

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Chapter 3 How does the Zetasizer μV work?

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.

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

Help Menu

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

Links to the Malvern website... including downloads and consumables.

Tip of the day... will give hints on how to use the Zetasizer software; a different

tip will appear each time this is selected. An option is given for turning off the Tip of the day dialogue that appears when the software starts.

About... gives details on the software version installed. Quote this if contacting Malvern Instruments.

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How does the Zetasizer μV work? Chapter 3



Toolbars

The toolbars contain a selection of tools that can be used to perform the most popular operations. Each tool will have its equivalent commands within the menu bar. For example, using the Open tool is equivalent to using the File-Open menu item.

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, or accessory, is not available it will be

shown “greyed out”.

Standard

Navigation

Folders

SOP Toolbar

Workspace

These tools perform the most popular File and Edit menu options functions.

Use the arrow icons to scroll up and down the records list. The Plate Navigator button is for viewing measurements that were made using a Zetasizer APS instrument. Greyed out for results made using a Zetasizer μV.

Selecting either of these will open the required folder - SOP, Measurement Data or Export - in a new window. This window is separate from the main application allowing the files to be copied, moved or deleted.

The SOP toolbar records the most recent SOPs used to enable quick selection. This list will also include any recent SOP player (.sopl) files. Refer to Chapter 9 for more information. The eject button is greyed out and reserved for use when using a Zetasizer APS instrument.

Allows selection and configuration of the Workspaces. Refer to Chapter 8.

The content and appearance of the toolbar can be changed using the ViewToolbars-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 5.

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Measurement file workspace

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 them 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.

If selected using the View menu, a Statistics bar will be available at the bottom of the record view. This can display the Standard deviation, the minimum, the maximum or the mean for the records selected.

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

Selecting a report tab will display a predefined report as selected by the Report pages tab in the workspace dialogues. Malvern supplies several reports that give different views of the measurement settings and results, whilst custom reports can be generated using the Report Designer.

Details on interpreting the reports can be found in Chapter 5.

 Title bar

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

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How does the Zetasizer μV work? Chapter 3



Measurement display

When a measurement is being performed a measurement display will appear.

The Measurement display shows the progress of the measurement. The screen display shown changes depending on the type of measurement being performed and the view tab selected.

 Status bar and Status icons

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

Customise.... dialogues to disable it.

On the right of the status bar, icons are shown to detail the operating state of the instrument and what additional features may be installed.

μV icon

Double-clicking the μV icon displays the Zetasizer μV model, serial number, software and firmware version of the instrument (only if the instrument is connected and switched on). If the instrument is disconnected from the software the icon will be greyed out:

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Chapter 3 How does the Zetasizer μV work?

21 CFR 11 icon

If the 21 CFR part 11 feature key is installed, the 21 CFR 11 icon will appear in blue on the right of the status bar.

If unavailable the icon will be ‘greyed’ out, like this:

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.

Units Conversion

A number of parameters within the software have the option to change the units used to define the parameter, i.e. Temperature has the option to use either ºC (Celsius) or K (Kelvin).

For all parameters where this option applies a scroll down list will be available alongside the parameter. The units can be selected in the SOP and other dialogues throughout the software.

The data is stored with a set of defaults, and is displayed with the selected units defined in the workspace chosen. This means that a particular record will be displayed with different units depending on the definition in the workspace used.

The Parameter unit chosen in the SOP dialogues will be displayed in all results and reports.

It is possible to subsequently alter the units used for the parameters in the Record and Report views - refer to Chapter 5.

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A Tutorial - Making a measurement

Introduction

After reading this chapter a user should be able to make simple measurements. The chapter goes through the basics from turning on the system to displaying the results of a measurement.

The first section, “Quick guide to making a measurement” will run through the basic steps, giving an overview of the measurement process. 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 in Chapter 10.

Manual and SOP measurements

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. Instead, alter an existing SOP and just change the required parameters.

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Chapter 4 A Tutorial - Making a measurement

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

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

Quick guide to making a measurement

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 laser

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 tempera-

ture 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

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

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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.

Note

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

Starting the Zetasizer software

Double click on the icon to start the software.

If the desktop icon is not available, select

Start-All Programs-Malvern Instruments-ZetasizerZetasizer

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. See Chapter 6 for more information.

Choosing the correct cell

Caution!

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

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

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Chapter 4 A Tutorial - Making a measurement

 Disposable polystyrene cuvettes are easily scratched and should never be used

more than once.

 Disposable 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.

Size measurements

Disposable polystyrene (DTS0012)

Typical solvent Water, Water/ethanol Water, most organic and

Optical quality Good to very good Excellent

Minimum Sample volume

Advantages Low cost

Disadvantages Not resistant to organic

Material Polystyrene Quartz

1ml 2μl

Single use disposable (no cleaning)

solvents

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

2 Microlitre Quartz Cuvette (ZMV1002)

inorganic solvents

Highest optical quality

Can use nearly any solvent

Low sample volume

Requires cleaning after measurement

Requires careful filling to avoid bubbles

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Disposable low volume polystyrene (ZEN0117)

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

Optical quality Good to very good Excellent

Minimum Sample volume

Advantages Low cost

Disadvantages Requires careful filling to

Material Polystyrene Glass

100μl 1ml

Low volume

Single use disposable (no cleaning)

avoid bubbles

Not resistant to organic solvents

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

Glass - square aperture (PCS1115)

Typical solvent Water, most organic and

inorganic solvents

Optical quality Excellent Excellent

Minimum Sample volume

Advantages Highest optical quality

Disadvantages Requires cleaning after

Material Glass Quartz

1ml 12μl

Can use nearly any solvent

Reusable

measurement

Glass - round aperture (PCS8501)

inorganic solvents

Highest optical quality

Can use nearly any solvent

Requires cleaning after measurement

Low volume quartz (ZMV1012)

Water, most organic and inorganic solvents

Highest optical quality

Can use nearly any solvent

Low sample volume

Requires cleaning after measurement

Requires careful filling to avoid bubbles

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Chapter 4 A Tutorial - Making a measurement

Low volume Glass flow cuvette (ZEN0023)

Typical solvent Water, most organic and

inorganic solvents

Optical quality Excellent Excellent

Minimum Sample volume

Advantages Highest optical quality

Disadvantages Requires cleaning after

Material Glass Quartz

75μl plus tubing 8μl

Can use nearly any solvent (tubing dependent)

measurement

With manual use requires careful filling to avoid bubbles

Low volume quartz (ZMV1008)

Water, most organic and inorganic solvents

Highest optical quality

Can use nearly any solvent

Low sample volume

Requires cleaning after measurement

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

Minimum Sample volume

Advantages Highest optical quality

Disadvantages Requires cleaning after

Material Glass Glass

1ml 1ml

Can use nearly any solvent

Reusable

measurement

Glass - square aperture (PCS1115)

Water, most organic and inorganic solvents

Highest optical quality

Can use nearly any solvent

Reusable

Requires cleaning after measurement

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Min. 10mm

Max. 15mm

Filling the cell

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

General advice

 Only clean cells should be used.

All size cells should be rinsed/cleaned with filtered solvent before use - see the Cleaning the cells section in the Essentials manual.

All Molecular weight cells should be rinsed/cleaned with the filtered standard (i.e. Toluene) or solvent, then dried in a dust free environment such as a laminar flow cabinet, before use.

 The cell should be filled slowly to avoid air bubbles from being created. Ultra-

sonication can be used to remove air bubbles - but only if the sample is suitable for use with ultrasonics.

 If using syringe filters for the solvent, never use the first few drops from the

syringe, in case there are any residual dust particles in the filter that may contaminate the solvent.

Size and Molecular weight measurements

Standard cells

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).

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

 When filling, tilt the cuvette and allow it to fill slowly (see above).

 To stop bubbles forming let the sample flow down the inside.

Note

When filled place a lid securely on the cuvette.

Low volume cell

This cell is designed to use the minimum volume of sample possible for a size or Molecular weight measurement. The sample must be pipetted carefully into the bottom of the cuvette, so it is filled from the bottom up.

The minimum volume that can be used is 12 microlitres. This will only partly fill the visible cell volume. After filling, carefully inspect the cell for trapped bubbles.

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Inserting the cell

In the status bar, the software will prompt when the cell needs to be inserted. This will always be after the SOP has been started - see the next section. When and how the cell is inserted will depend on the application, and the measurement choices selected.

Size and Molecular weight measurements

For these measurements, do the following:

1. Press down on top of the cell area lid  (where it is marked “Push”) to unlock

the catch, and then release

. The lid will rise to an upright, open position.

Hold the cell gently between thumb and finger, being careful to only hold the top part of the cell, and lower it into the cell holder until resistance is felt.

Some cells have opaque surfaces as well as polished optical surfaces. A polished

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Chapter 4 A Tutorial - Making a measurement

optical surface must be inserted so that the “light path” indicator arrow in the cell enclosure passes through it - i.e. facing the front of the instrument. Most cells have a small triangle at the top to indicate the side that faces the front. This is especially critical for Molecular weight measurements.

If a flowcell is used, insert the sample tubes into the threaded inserts and screw into the top of the flowcell. The tubing is then inserted into the channel on the side of the cell area.

2. Close the lid, pressing firmly where it is marked “Push” to secure the catch.

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A Tutorial - Making a measurement Chapter 4

Making an SOP measurement

If a measurement is being made using an SOP, then all the hard work has already been done. The instrument has been turned on and the software started; the sample has been prepared and added to the cuvette. 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 is used for all the measurement records (not recom-

mended).

 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.

To open an existing measurement file:

1. Select File-Open-Measurement File or .

2. A dialogue will appear allowing selection of a measurement file.

3. Select Open.

To create a new measurement file:

1. Select File-New-Measurement File or .

2. A dialogue will appear allowing the new measurement file to be named and

specify where it will be saved.

3. Select Save.

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Chapter 4 A Tutorial - Making a measurement

Note

All measurement files have the extension .dts. This is added automatically to all new files.

Starting an SOP measurement

Everything should now be ready to make the actual measurement.

To start an SOP measurement, select Measure-Start SOP. The Open SOP dialogue will appear. Select the SOP that will be used and select Open. If an SOP has not been specified for the sample, read Chapter 9 for details on how to create one.

Pre-measurement instructions may appear advising of any actions that need to be performed before the measurement can proceed. Pressing Sample in the left column of the dialogue will open the Sample dialogue, allowing the measured sample to be named. 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 Sample dialogue. If the dialogue does not appear, but is required, select the Settings button in the measurement display.

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 two 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 with new record being shown in the measurement file window. The results can now be viewed - see Displaying the results in Chapter

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A Tutorial - Making a measurement Chapter 4

Making a manual measurement

Making a manual measurement is essentially the same as making an SOP measurement, except that, where as in an SOP measurement all the measurement options are pre-specified, here it will be necessary to set them immediately.

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

The dialogues are virtually identical to those in the SOP editor when used to define a new SOP, please refer to Chapter 9.

Once all settings have been made, if required, select the Save as... icon or File-

Save as... from the menu to store the settings. Click the OK button to close the Manual measurement editor and return to the measurement display.

Note

The manual measurement settings can be viewed and subsequently saved by selecting Edit-Extract SOP.

The Measurement display

1 4

When an SOP or manual measurement is started the measurement display is displayed, 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

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Chapter 4 A Tutorial - Making a measurement

sequence. The dialogues displayed depend upon the measurement type selected. The diagram below shows the display for a size measurement.

The features of the measurement display are:

 Button bar

The button bar provides the control for the measurement operation.

The Settings button opens the measurement settings dialogue. Extra comments and changes to the measurement parameters can be added prior to the measurement being started.

These Start and Stop 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. When Stop is pressed, a warning box will appear asking “Are you sure you wish to abort the measurement”.

Opens the Help file.

The Accessory button is greyed out - only available for Zetasizer Nano instruments.

When the Start button is pressed the Manual control dialogue will be closed.

 Status bar

The status bar shows instructions and the current operation in the measurement sequence.

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A Tutorial - Making a measurement Chapter 4



Progress meter

The progress meter shows how far the measurement has progressed plus the number of measurements performed and the measurement runs completed. Also shown are the temperature, measurement position and attenuator settings.

 Tab views

The Tab views enable the progress and results of the measurement to be viewed. The first tab shows the results, and will change with respect to the measurement type and results view selected. In the above example, this tab is labelled Intensity PSD to identify that intensity results are being viewed. This tab shows different graph plots relevant to the measurement type selected. The other three tabs dialogues - Multi-view, Log sheet and Expert advice - are standard for each Measurement type.

 The views displayed in the first (result) tab, can be altered by right-clicking on

the graph and selecting from the list displayed. A graph only, or a graph with a summary table will be shown depending upon the view chosen. The Result tab is named after the result view chosen.

 The Multi-view tab displays the results in three smaller windows. As with the

first (result) tab, the view in each separate window can be altered by right-

clicking in the window and selecting a different view from the list displayed. The size of each window can altered by moving the borders around.

The Tab views for each measurement type are described below.

Size measurements

Result tab (1st tab)

The result tab will show the result obtained as the measurement progresses. The result view will be updated after every run of the measurement. The result shown will be the sum of the acceptable data collected.

The Result tab is named after the result view chosen, the default result view shown is Intensity PSD. Different views can be selected by right-clicking on the graph and selecting from the list displayed. A graph only, or a graph with a summary table will be shown depending upon the view chosen.

The views available are: Count Rate, Correlation Function, Intensity PSD, Volume PSD and Number PSD. These are described below.

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Normal count rate

Count rate (Kcps)

 Count rate

Displays the number of photons detected per second. The count rate is useful for monitoring the sample quality.

Normal count rate display.

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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)

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Thermal gradients

ount rate (Kcps)

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Aggregating sample Sedimenting sample

ount rate (Kcps)

Time

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A Tutorial - Making a measurement Chapter 4

Small size sample

Contaminated sample

 Correlation function

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

Large size sample

g(2)-1

Time Time

g(2)-1

Time Time

g(2)-1

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Noisy data = variable results

g(2)-1

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 Intensity PSD

Displays the result as an Intensity based Particle Size Distribution. A summary table will displayed alongside the graph.

 Volume PSD

Displays the result as a Volume based Particle Size Distribution. A summary table will displayed alongside the graph.

 Number PSD

Displays the result as a Number based Particle Size Distribution. A summary table will displayed alongside the graph.

Multi-view Tab

The Multi-view enables three different result views to be shown at the same time. The view in each separate window can be altered by right-clicking in the window and selecting a different view from the list displayed. The same views as used in the result tab are available.

Log sheet Tab

This displays the progress of the measurement. The log sheet can be saved by rightclicking the mouse on it, pressing the Save to file... button that appears and saving it as a .txt file.

Expert Advice Tab

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The Expert Advice tab reports on how good the quality of the measurement is. This will show how good the measurements are and whether they display any unwanted attributes, such as aggregation.

Chapter 4 A Tutorial - Making a measurement

Refer to the Expert Advice section in Chapter 10 for a description.

Molecular weight measurements

Result tab (1st tab)

Please also refer to size description above. For Molecular weight measurements the default result view shown is a Debye plot. The Different views that can be selected are described below: Intensity PSD, Count Rate, Correlation Function, Volume PSD, Number PSD and Debye.

 Intensity PSD

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. Please refer to the Size description above.

 Count rate

Please refer to the Size description above.

 Correlation function

Please refer to the Size description above.

 Volume PSD

Please refer to the Size description above.

 Number PSD

Please refer to the Size description above.

 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.

Multi-view Tab

This is as the Size description above, except for Molecular weight specific view choices.

Log sheet Tab

Please refer to the Size description above.

Expert Advice Tab

Please refer to the Size description above.

Flow-mode measurements

Result tab (1st tab)

The result tab will show the result obtained as the measurement progresses. The result view will be updated after every run of the measurement.

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The Result tab is named after the result view chosen, the result view shown above is Flow trace vs Time. Different views can be selected by right-clicking on the graph and selecting from the list displayed.

The views available are: Count Rate, Correlation Function, Flow trace vs Volume and Flow trace vs Time and Monitor.

 Monitor enables the count rate signals to viewed before a measurement is run.

Multi-view Tab

As the Size description above, except for Flow-mode specific view choices.

Log sheet Tab

Please refer to the Size description above.

Expert Advice Tab

Please refer to the Size description above.

Trend measurements

The trend dialogues are generally the same as those used when either a size or Zeta measurement SOP is chosen. The difference is the inclusion of an extra Trend graph view - this will show an evolving plot as the measurement progresses. Melting Point measurements can be performed using the Trend measurement type.

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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.

The paragraphs that follow describe the main measurement types the Zetasizer μV performs: Size and Molecular weight.

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.

As soon as a run is completed, a preliminary size result will be 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 measurements, the sequence has been described below:

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A Tutorial - Making a measurement Chapter 4

 Press Start to begin the dark count measurement.

The laser is turned off and a measurement is taken of the background light level.

 Insert the scattering standard cell (i.e. Toluene) and press Start when ready.

The measurement will measure the scattering intensity of the scattering 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 will then continue.

 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.

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Records and Reports

- Viewing the Results

Introduction

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

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

Displaying the results

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

Note

The Records view parameters and Report tabs that are displayed are dependent upon the Workspace settings selected- refer to Chapter 8.

Records view

Once the measurement is complete, a new measurement record will be added to 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 Tools-Settings- Configure Workspace dialogues, the record view shown below depicts the default summary workspace (see Chapter 8).

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Chapter 5 Records and Reports - Viewing the Results

Melting point records

To view a sequence of Melting point records, select the first record in the sequence; all subsequent records in that sequence will then automatically be selected.

Outliners

For Melting point records, outliners are employed. This enables the main ‘parent’ record to be shown without the ‘child’ records also being displayed.

Selecting will display the ‘child’ records, selecting will hide them.

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Records and Reports - Viewing the Results Chapter 5

The Outlined record view can be turned off and on by selecting View-View typeSorted or Outlined or by right-clicking the mouse on the table, as shown above.

Statistics bar

Use this to display the Standard deviation, the Relative Standard deviation, the minimum, the maximum or the mean of a selected number of records. Select View

-Statistics bar from the menu bar, and then the options to display. A bar will be added to the bottom of the record view displaying each of the parameters selected.

To display the statistics of a number or records, hold down Shift or Ctrl and then the records required in the normal Windows fashion; the statistics bar will be automatically updated as each record is chosen.

View-Statistic bars-Hide will remove the statistics bar from the record view.

Units conversion

Within the Records view, it is possible to change the units used to define some of the parameters. If the option is available, clicking on a parameter column will

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Chapter 5 Records and Reports - Viewing the Results

display a parameter icon enabling the units to be changed. A check mark (9) will be displayed next to the currently selected units.

Note

Changing the units of a parameter will alter the units for each occurrence of the parameter in any report then displayed.

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.

To view a sequence of Melting point or Titration records, select the first record in the sequence; all subsequent records in that sequence will then automatically be selected.

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

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 the different aspect ratios of the printed page and the computer screen.

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.

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Records and Reports - Viewing the Results Chapter 5

Note

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

Units conversion

As in the Records view, within the results section of each report, it is possible to change the units used to define some of the parameters displayed. If the option is available, right-clicking on a parameter will display a parameter icon enabling the units to be changed. A check mark (9 ) will be displayed next to the currently selected units.

If present, right-clicking on the table will also bring up the option to change the units displayed. For a table, select units from the displayed menu.

Note

Changing the units of a parameter in any field of the report, will alter the units for every other occurrence of the parameter in the report, and the parameter column in the Records view, however the default unit in the record is not changed.

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Chapter 5 Records and Reports - Viewing the Results

Size measurements - standard report

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

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

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 Sample details

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

 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.

 Duration used(s)

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Duration of measurement, used in the analysis of the result.

Records and Reports - Viewing the Results Chapter 5

Ln G1[] abtct2dt3et4……++ + + +=

 Cell type

This displays the cell type selected.

 Results

This section gives the results of the measurement. The values given here will be based either on intensity, volume or number, depending on which view tab is selected.

The results section gives three pieces of information:

 Z-average Size (also known as the “cumulants mean”)

In Dynamic Light Scattering (DLS) 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 solvent.

In any other case, the Z-average size can only be used to compare results with samples measured in the same solvent, by the same technique.

The cumulants analysis only gives two values, a mean value for the size, and a width parameter known as the Polydispersity, or the Polydispersity 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 G

correlation function.

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 solvent 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).

 PdI

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 and 22412.

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Chapter 5 Records and Reports - Viewing the Results

 Intercept

This is the amplitude of the G

correlation function at time 0. For a good meas-

urement it will be 0.85 to 0.95.

Lower values will be obtained for measurements on weaker scattering sample, or in turbid sample where background scattering is significant. Values >1 will only occur for samples with dust that makes the sample fluctuate wildly.

 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.

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

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Records and Reports - Viewing the Results Chapter 5

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,

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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.

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.

Other Size reports

Other Malvern views available for size measurements include:

 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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Chapter 5 Records and Reports - Viewing the Results

 Size quality report (M)

This report is described below.

Size quality report (M)

This is a summary report detailing whether the result meets the Malvern Instruments quality criteria for a size measurement. The report indicates Result meets quality criteria, or Result does not meet quality criteria in which case a list of parameters that do not meet the criteria, and probable reasons will be displayed. Some of these parameters will only be displayed if the research software has been used for the measurement.

The parameters, followed by probable reasons displayed are:

 z Average is smaller than lower size display limit

Wrong size limits used in display range

 z Average is larger than upper size display limit

Wrong size limits used in display range

 z Average is smaller than lower size analysis limit

Wrong size limits used in analysis range

 z Average is larger than upper size analysis limit

Wrong size limits used in analysis range

 Polydispersity index is very high

Sample is very polydisperse and may not be suitable for DLS measurements Sample contains large particles/aggregates/dust Wrong measurement position selected

 Correlation function intercept out of range

Sample concentration too high (multiple scattering) Sample concentration too low Sample fluorescence Sample absorbance (coloured samples) Wrong measurement position selected

 Check first correlation point selection for multimodal analysis

 In range figure is low

Presence of large or sedimenting particles Sample fluorescence Sample absorbance (coloured samples)

 Count rate is out of range (too low)

Attenuator not set to automatic Sample concentration too low Sample absorbance (coloured samples) Sample is not stable during measurement

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Records and Reports - Viewing the Results Chapter 5

 Count rate is out of range (too high)

Attenuator not set to automatic Sample contains large particles/aggregates/dust Sample is not stable during measurement

 Insufficient signal collected

Measurement duration not set to automatic Filter factor in research software not set to default (50%)

 Cumulant fit error high

Data quality too poor for cumulant analysis Sample too polydisperse for cumulant analysis Inappropriate cumulant analysis settings in Research Software

 Multimodal fit error high

Data quality too poor for distribution analysis Sample too polydisperse for distribution analysis Inappropriate distribution analysis settings in Research Software

 Check cumulants and distribution fit errors

Sample too polydisperse for cumulant analysis - suggest rely on distribution analysis

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Chapter 5 Records and Reports - Viewing the Results

Molecular weight measurements - standard report

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

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

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,

Sample and measurement details

These two sections show measurement details and details of parameters relating to the sample. Please see the size description for details.

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.

 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

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shape; i.e. sphere, coil, cylinder or no shape correction.

Records and Reports - Viewing the Results Chapter 5

 System details

The System section gives details on settings configured during the measurement process. Please see the size description for details about Temperature, and Cell type.

 Results

The results section gives four pieces of information:

 Molecular weight

Shows the measured weight of a molecule within the measured sample expressed in atomic mass units; indicated in KiloDaltons (KDa).

 Shape correction

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

 Second Virial Coefficient (A

)

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.

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Chapter 5 Records and Reports - Viewing the Results

Flow-mode measurements - Standard report

The Standard Flow-mode report, Chromatogram Summary (M), gives the same information as seen in a standard size report, plus additional information relating to the flow duration and rate used.

The three main peaks in the measurement will also be shown, displaying the sample intensity, width, Molecular weight and start and end flow volumes.

Additionally a the flow trace result graph will be displayed. This can be viewed either in Time or Volume:

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Records and Reports - Viewing the Results Chapter 5

Trend measurements (including Protein Melting point)

Trend measurements can be viewed using the reports: Trend, Trend v Custom 1 and Trend v Custom 2 reports. Melting point trend measurements can be viewed using the Melting point (M) report.

A trend report gives the same information as seen in a standard size or Zeta report, plus a graph showing the trend progression.

Additionally a melting point report will show 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 achieved.

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Chapter 5 Records and Reports - Viewing the Results

Page 5-16 MAN 0428

Sample Preparation

Introduction

Before filling the cell or cuvette that will be used in the measurement, the sample will need to be prepared. To ensure reliable and accurate measurements proper sample preparation is extremely important.

Preparation of the sample for the different measurement types will involve specific preparation techniques. 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 or molecular 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).

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

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Chapter 6 Sample Preparation

Considerations for small particles

Minimum concentration

For particle sizes smaller than 10nm, the major factor in determining a minimum concentration is the amount of scattered light that the sample generates. In practice, the concentration should generate a minimum count rate of 10,000 counts per second (10kcps) in excess of the scattering from the solvent. As a guide, the scattering from water should give a count rate in excess of 10kcps.

Filtration

All liquids used to dilute the sample (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 solvent.

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 sample. Limits for the use of ultrasonication in terms of intensity and application time are strongly sample dependent. Some materials can even be forced to aggregate using ultrasound.

Emulsions and liposomes should not be ultrasonicated.

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 laminer 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.

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Sample Preparation Chapter 6

Increasing sample concen tration

Solvent

1 2

Intercept point

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

3 4

excluded.

ill 6766

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

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

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ill 6767

Concentration

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 APD detector used in the instrument has a linear operating range. Therefore the maximum sample concentration should give a count rate of less than 1000Kcps.

Chapter 6 Sample Preparation

Count rate meter

After preparing the sample, the count rate can be checked by using the count rate meter. Selecting Tools-Count rate meter will display the following dialogue.

The Count rate is displayed in Kcps (Kilo counts per second), and enables the sample quality and concentration to be checked without having to start a measurement.

Count rate monitor

By inspecting the count rate monitor the quality of the sample can be observed. Selecting the Auto Scale check box will automatically scale the graph as the count rate monitoring progresses. Reset will delete the current display.

 Normal count rate

The monitor will show a relatively stable count rate.

 Dust in the sample

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 unless all measurement runs contain spikes from contaminants.

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Sample Preparation Chapter 6

 Drifting count rate

This may indicate that thermal gradients are present in the sample, and further time is required for temperature equilibration.

 Increasing/Decreasing count rate

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

In addition to displaying the count rate, the dialogue allows changes to the cell and attenuator position settings. For normal operations both will be at the default setting.

Attenuator

The Attenuator setting can be used to monitor the scattering intensity without having to start a measurement.

 To change the attenuator position, alter the value using the slider bar.

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Chapter 6 Sample Preparation

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Part 2 Supervisor’s Guide

Security

Introduction

The systems may be used by users of varying skill levels. With the Zetasizer it is possible to limit user access so that certain functions such as editing SOPs, deleting records and editing results can be reserved for specific users only.

Within the Malvern Security system, one or more persons are set up as the administrator. The administrator then controls access to the instrument by defining “User groups” and “Permissions”.

 A User group is one or more persons that have the same access rights.

 Permissions are the access rights that are allowed for each user group, these

will range from allowing SOPs to be edited to disabling view selection.

The administrator adds operators to a group and assigns each operator a password. Each operator’s identification and password are entered to enable access to the software.

The first time the software is run the security system will be disabled and an administrator user and administrators group will be created by the system. This is so at least one user will have permission to configure the security system.

Note

The Malvern security system can be upgraded to 21 CFR part 11 compliance by installing a “Feature key”. Once this is installed, 21 CFR part 11 security settings can be applied and ‘Audit trails’ can be viewed. If the feature key has been installed, the grey 21 CFR 11 icon on the status bar will turn to yellow. Please note that this manual will not detail the 21 CFR part 11 option but will concentrate only on the standard security software.

The first task is to set up the administrator account.

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Chapter 7 Security

Initial start-up - set up the administrator

Note

It is vital that at least one user has access to the configuration of the security system.

Select Security-Configure Security.

The first time the system is run, there will be just one user (Administrator) and one group (Administrators). This group is originally set to only allow configuration of the security system and to deny access to all other features of the system.

On first start up of the software the security system defaults to a member of the Administrators group with no password.

To secure the system, a password must first be specified for the administrator account (detailed later) and then the security enabled using the Options-Security settings... menu.

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Security Chapter 7

Note

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

Remember that after any changes to the security system - adding users, changing of permissions, etc. The new changes must be saved (User-Save). A dialogue will appear on exiting to give a reminder on saving changes.

Enabling security

Within the Security configuration dialogue select Options-Security settings and select the Enable security check box.

With security enabled each user must login when the software starts. Once logged in only their relevant permissions will be accessible, all other functions will be greyed out.

Note

With the 21 CFR part 11 feature key installed a different dialogue will be displayed. Once 21 CFR part 11 security is enabled it cannot be disabled.

To change operators when the software is already open select Security-Logout and then Security-Login and enter the appropriate passwords.

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Chapter 7 Security

User groups

Note

Only users assigned to the Administrator group can add or edit the user group properties.

Adding/editing a group

Select Security-Configure security to enter the Security configuration dialogue. Select User-New Group... to display the Group Properties dialogue, alternatively double-click on an empty row. When editing double-click on an existing group to display the dialogue.

Enter a Group name and a Description of the group’s purpose. Example names might be:

 Operators - General users of the system.

 Supervisors - Skilled operators responsible for configuration and SOP crea-

tion.

 Administrators - Users authorised to configure the security system.

The Members’ section of the dialogue shows a list of all the users currently assigned to the group. To add a user press Add and a list of all users not currently

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Security Chapter 7

allocated to that group will be shown. Selecting one or more users (hold down the control key to select multiple users) and press OK to add those users to the group.

To remove a member, simply select the member and press the Remove button. Note that members are only removed from the group, they are not removed from the system.

The Permissions section of the dialogue allows functions of the software to be enabled/disabled for the group. To set the access permissions for the group, simply scroll down the list of permissions and check the boxes for the permissions required. This can also be done with a mouse or by highlighting a permission and pressing the space bar. The permissions list can be navigated using the up and down keys.

Users

Note

Only users assigned to the administrator group can add or edit user properties.

Adding/editing a user

Select Security-Configure security to enter the Security configuration dialogue. Select User-New User... to display the User properties dialogue, alternatively double-click on an empty row. When editing double-click on an existing user to display the dialogue.

Each user must have a unique Username. This, along with the user Password, forms the unique key required to identify each individual using the system. The

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Chapter 7 Security

Username is commonly an abbreviated form of the individual name or a unique identifier such as an employee code.

The Full Name is used to hold the full printed name of the individual and can be used on reports to identify the user if an employee code is used as a Username.

The Description field is optional and is just used to add some descriptive text to the user details.

Generally it will be the administrator entering these details. The administrator can enter the password but this would require the administrator to know a users password beforehand. A more secure approach is for the administrator to specify a previously arranged password, such as the user’s name, and force the user to change their password the next time they log on using the password check boxes.

Press the Groups button to allocate the user to the required groups. Use the Add and Remove buttons to allocate the group(s) appropriate.

Password options

 User must change password at next logon

The change password dialogue will be shown when the user next logs in, allowing the user to change their password. This allows the security administrator and a user to set up a user account without the administrator knowing the users final password.

Users can change their own passwords once logged in by using the Security- Change password menu.

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Malvern Zetasizer uV User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual