Malvern Zetasizer Nano Series Accessories Manual
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Malvern Zetasizer Nano Series Accessories Manual

Malvern Zetasizer Nano Series Accessories Manual

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  • Page 1 Zetasizer nano series Malvern...
  • Page 3 Zetasizer Nano accessories guide MAN0487 Issue 1.1 April 2013...
  • Page 4 United Kingdom. Tel + [44] (0)1684-892456 Fax + [44] (0)1684-892789 Zetasizer, Malvern and the 'hills' logo are registered trademarks in the UK and/or other countries, and are owned by Malvern Instruments Ltd. NIBS and M3-PALS are trademarks of Malvern Instruments.

  • Page 5: Table Of Contents

    T able of contents Introduction and accessory range ......... 1-1 Introduction .
  • Page 6 Table of Contents Zetasizer Nano accessories guide ..... 4-14 The Flow-mode measurement display ......4-15 Displaying the flow-mode report Microrheology .

  • Page 7: Introduction And Accessory Range Introduction

    Introduction and accessory range Introduction This manual give an overview the accessories that are available for use with the Zetasizer Nano series of instruments. This manual is a supplement to the following manuals: Zetasizer Nano user manual  Zetasizer Nano basic guide ...
  • Page 8 Chapter 1 Introduction and accessory range Cells and Cuvettes Zeta potential measurements DTS1070 Folded capillary cell Maintenance-free capillary cell primarily designed for zeta potential measurements. (This cell is a direct replacement for DTS1060/61). ZEN1002 Dip cell Cell used to provide repeatable measurements of aqueous, and non-aqueous samples.
  • Page 9 Introduction and accessory range Chapter 1 Cell to Zetasizer Nano instrument compatibility table The table below indicates which cells and cuvettes are compatible with which instruments of the Zetasizer Nano range. Cell Zeta Size ZS90 DTS1070 • • • • •...
  • Page 10 If fitted the Zetasizer Nano instrument label will include an ‘HT’ identification and an option part number label will be attached to the front of the cuvette holder. ZEN9063 Extends the upper temperature range of the Zetasizer Nano series  ...
  • Page 11 Introduction and accessory range Chapter 1 Narrow band filter ZEN9051 Narrow band filter for ‘Green’ badged Zetasizer Nano S instruments ZEN9052 Narrow band filter for ‘Green’ badged Zetasizer Nano ZS instru- ments ZEN9061 Narrow band filter for ‘Red’ badged Zetasizer Nano S, Z and S90 instruments ZEN9062 Narrow band filter for ‘Red’...
  • Page 12 Chapter 1 Introduction and accessory range Page 1-6 MAN 0487...

  • Page 13: General Cells And Cuvettes

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

  • Page 14: Cuvette Holder

    When not being used, place these in the holder to the left of the tray. The cuvette holder includes a serial number, model number and option labels. These identify the instrument and should be quoted in any correspondence with Malvern Instruments. Page 2-2 MAN 0487...

  • Page 15: Cell And Cuvettes

    Surface zeta potential (SZP) cell Zeta potential All the cells mentioned are available from Malvern and should be used with the supplied cell caps. Using the caps will ensure greater thermal stability of the sample, as well as preventing dust introduction and possible spillage.
  • Page 16 Chapter 2 General cells and cuvettes Cell and cuvette options The cells and cuvettes described in this section can be used for all measurements. Folded capillary cell Disposable polystyrene (DTS1070 / DTS1060/61) (DTS0012) Application Size, zeta potential Size Typical solvent Water, water/alcohol Water, water/ethanol Optical quality...
  • Page 17 General cells and cuvettes Chapter 2 Disposable low volume Disposable low volume polystyrene (ZEN0118) polystyrene (ZEN0040) Application Size Size Typical solvent Water, water/alcohol Water, water/alcohol Optical quality Good to very good Good to very good Minimum 50μl 40μl Sample volume Advantages Low cost Low cost...
  • Page 18 Chapter 2 General cells and cuvettes Low volume Glass flow Low volume quartz cell (ZEN0023) (ZEN2112) Application Size Size Typical solvent Water, most organic and Water, most organic and inorganic solvents inorganic solvents Optical quality Excellent Excellent Minimum 75μl plus tubing 12μl Sample volume Advantages...
  • Page 19 General cells and cuvettes Chapter 2 Use, cleanliness and filling advice Note Before filling and using a cell or cuvette, consult the cleaning section for each cell or cuvette, and perform any cleaning and maintenance proce- dures described. When filling the cell there are several actions to consider; some that apply to all cells and others that are only applicable to the measurement type and the cell chosen.

  • Page 20: Size And Molecular Weight Cuvettes

    Chapter 2 General cells and cuvettes Size and molecular weight cuvettes Filling a cell or cuvette Fill the cell with the prepared sample as described below. Also refer to the filling advice given earlier in this chapter. Standard cuvettes 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.
  • Page 21 General cells and cuvettes Chapter 2 Note When filled place a lid securely on the cuvette. Low volume cuvettes These cells are 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.
  • Page 22 Chapter 2 General cells and cuvettes Small triangle towards button To Autotitrator Flowcell connections ill 8510 Flowcells - using the MPT-2 Titrator Follow these instructions for connecting a flowcell when using the MPT-2 Titrator. Always minimise the tubing within the cell area before inserting into the pinch valve channel.
  • Page 23 General cells and cuvettes Chapter 2 The tubing is attached to the flowcell using threaded inserts; push the sample  tubes into the inserts and screw into the top of the flowcell. The tubing is then inserted into the pinch valve channel; push both tubes ...

  • Page 24: Folded Capillary Cell

    This removes the chances of cross- contamination. The cells are inserted with either the Malvern logo (DTS1070) or the weld line (DTS1060/61) facing the front of the instrument - refer to the Inserting the cell section later in this chapter.
  • Page 25 General cells and cuvettes Chapter 2 Filling a folded capillary cell The folded capillary cells should be filled with the prepared sample as described below. Both cells should be rinsed/cleaned with filtered dispersant before use; refer to Cleaning the folded capillary cell later in this section Note Filling a cell for a protein mobility measurement involves a different technique.
  • Page 26 Chapter 2 General cells and cuvettes Turn the cell upright and continue to inject slowly until the sample is reaches  the fill area as shown . Fill between shoulder of cell and the FILL MAX line. Check again for bubbles in the cell. Tap the cell gently to dislodge these. ...
  • Page 27 General cells and cuvettes Chapter 2 Check no air bubbles form in the cell. Tap the cell gently to dislodge any that  do form. Turn the cell upright and continue to inject slowly until the sample is at the top ...
  • Page 28 Chapter 2 General cells and cuvettes Note The stoppers must be fitted before a measurement is performed. Ensure that one stopper is fitted firmly, and the other one loosely, to avoid pressur- isation of the cell. Inserting the folded capillary cell ill 8507 Place a thermal contact plate into the recess on either side of the folded cap- ...
  • Page 29 The diagram below shows the cell with the preferred orientation in the cell holder. DTS1070 DTS1060/61 ill 8798 / 7945 DTS1070 cell When inserting the cell, ensure that the Malvern logo faces towards the front of the instrument. Press down until the cell clicks into place. Zetasizer Nano accessories guide Page 2-17...
  • Page 30 Chapter 2 General cells and cuvettes DTS1060/61 cell The cell is oriented such that the weld line is towards the front of the instrument. Press down until the cell clicks into place. The cell is made of two different parts (front and back part), welded together. Tests indicate that inserting the cell with the front part towards the laser gives better count rates, and hence this is the preferred cell orientation.
  • Page 31 General cells and cuvettes Chapter 2 Cleaning the folded capillary cell This cell is intended to be used once then discarded. We recommend that, before a cell is used for the first time, it is flushed through with ethanol or methanol to facil- itate wetting.

  • Page 32: Dip Cell

    Chapter 2 General cells and cuvettes Dip cell (ZEN1002) Description The Dip cell is used to provide a method to measure the zeta potential of both aqueous and non-aqueous samples. A number of samples can be prepared and the Dip cell inserted to measure each one in turn.
  • Page 33 General cells and cuvettes Chapter 2 To ensure a minimum sample volume is provided for the sample to be measured but protect against overfilling, we recommend the cuvette is filled to a depth of between 7mm and 10mm (before the Dip cell is inserted). The minimum level relates to approximately 0.7ml of sample.
  • Page 34 Chapter 2 General cells and cuvettes 10mm 45° Front Coloured band Front Triangle Front ill 8508 Note With the procedure complete, the measurement face of the cuvette (some have a small triangle at the top of the cell) and the coloured band on the Dip cell label must face in the same direction.
  • Page 35 General cells and cuvettes Chapter 2 ill 8509 Place the Dip cell immediately into an empty cuvette for storage.   This will prevent any potential damage occuring either to the cell electrodes or the workspace. Remove the sample cuvette afterwards and place in the cuvette holder ...
  • Page 36 Note The electrode holder is made from Natural PEEK (Polyetheretherketone) which is resistant to a wide range of chemical products. However, seek advice from Malvern and the sample manufacturer before using strong acid or base. Chemical compatibility - Dip cell With proper use, only the central electrode section of the Dip cell will ever come in contact with sample.

  • Page 37: High Concentration Cell

    General cells and cuvettes Chapter 2 High concentration cell (ZEN1010) Description The High concentration cell is intended primarily for the measurement of zeta potential of concentrated aqueous samples. The cell can be used in conjunction with the MPT-2 for automated titrations. The cell consists of a high precision optical measurement block held within electrode chambers.
  • Page 38 Chapter 2 General cells and cuvettes ill 8448 Inserting the High concentration celI The High concentration cell is inserted into the instrument and connected to the Titrator in the same manner as the Folded capillary cell. ill 8449 The metal face of the cell must face the front of the instrument; this is to ensure good thermal contact between cell and instrument.
  • Page 39 General cells and cuvettes Chapter 2 External surfaces of the assembled cell can be wiped clean with a weak soap solu- tion. Intensive cleaning The cell first has to be disassembled before cleaning can be performed. ill 8447 Remove the screw cap . ...
  • Page 40 Chapter 2 General cells and cuvettes Component Cleaning method Screw cap Wipe clean with a mild soap solution Outer casing Black part of casing (Rear - Delrin): Wipe clean with a mild soap solution. Metal part of casing (Front - Stainless steel): Immerse the casing in Hellmanex and place in a gentle ultrasound bath (30 Watts) for five to 15 minutes.
  • Page 41 General cells and cuvettes Chapter 2 Surface zeta potential cell (ZEN1020) Description The surface zeta potential cell is intended for the measurement of the zeta potential at the surface of a flat material in an aqueous environment. The cell is a dip cell type device an be used with 1ml cell DTS0012 and PCS1115.
  • Page 42 Chapter 2 General cells and cuvettes Page 2-30 MAN 0487...

  • Page 43: Surface Zeta Potential Cell

    Surface zeta potential cell Introduction This chapter gives an overview of the Zetasizer Nano cell for measuring surface zeta potential. It describes how to use, insert and clean the cell to ensure reliable and consistent measurements. The Surface zeta potential (SZP) cell is intended for the measurement of the zeta potential at the surface of a flat material in an aqueous environment.

  • Page 44: Surface Zeta Potential Cell Introduction

    Chapter 3 Surface zeta potential cell The cell is supplied with the following components to prepare, load and set the sample: Surface zeta potential cell with A 12-well plate for storing the samples palladium electrodes 10 PEEK sample holders A screwdriver for cell tightening Forceps for sample handling A cell height alignment tool and a sample holder for gluing the sample to...

  • Page 45: Measurement Technique

    Surface zeta potential cell Chapter 3 Measurement technique A surface zeta potential measurement consists of attaching a sample to a mount or holder that is then held in place between two electrodes. The sample is then immersed in an appropriate aqueous solution, containing tracer particles. The apparent tracer mobility is now measured at a number of different distances from the sample surface.

  • Page 46: Preparation For Measurement

    Chapter 3 Surface zeta potential cell Preparation for measurement Before a measurement can be performed the cell must first be loaded onto the sam- ple holder and then attached to the cell. The complete cell is then inserted into a standard cuvette and placed into the instrument.
  • Page 47 Surface zeta potential cell Chapter 3 The sample is then attached to the sample holder using an appropriate adhesive such as Araldite (refer to note overleaf for guidelines on glue selection). A sample gluing tool is provided to hold the sample holder during the gluing ...
  • Page 48 Chapter 3 Surface zeta potential cell ill 8690 Insert the cell assembly into the tool, so that the white mark on the cell is facing the front of the tool, indicated by the white spot, and tilting forward. Adjust the cell cap to alter the sample barrel position until the surface of the sample is aligned with the zeroing target on the tool window.
  • Page 49 Surface zeta potential cell Chapter 3 The sample barrel position should be adjusted so that the sample surface and the centres of the two zeroing targets all line-up exactly. With the sample height set, the cell cap also needs to be zeroed. Loosen the cap screw, then rotate the cell cap until the white mark on the cap is in line with the white mark on the cell body.
  • Page 50 Chapter 3 Surface zeta potential cell The cuvette must not be filled more than the recommended maximum depth  of 20mm before insertion of the cell . Tilt the cuvette to a maximum angle of 45° . This is to avoid spilling the dis- ...
  • Page 51 Surface zeta potential cell Chapter 3 Hold the base of the SZP cell cap and the top of the cuvette simultaneously .  Ensure the white marks  on the cap and cell body, and the cuvette triangle , are facing the front of the instrument and push the cell into the cell holder until it stops - a 'stop' on the surface potential cell must rest on the top of the cell holder.

  • Page 52: Controlling An Szp Measurement Via An Sop

    Chapter 3 Surface zeta potential cell Controlling an SZP measurement via an Once the cell has been inserted into the Zetasizer and zeroed, a measurement can be made. This is done using an SOP, or a manual measurement in the usual manner.
  • Page 53 Surface zeta potential cell Chapter 3 Cell Please refer to the Sample - Cell description in the size SOPs section of the main user manual. As the surface zeta potential cell was selected as the measurement type, this cell will be the only cell choice available.
  • Page 54 Chapter 3 Surface zeta potential cell Settings Description The SZP (Surface zeta potential) measurement duration measurement options are the same as standard measurement duration duration options available during normal zeta potential measurements. The SZP measurements options define the number of measurements repeat measurements made at each displacement away from the surface and the length of any delay between repeat...
  • Page 55 Surface zeta potential cell Chapter 3 Tracer measurement The Tracer measurement SOP window is similar to the standard zeta potential Measurement window. Where appropriate please refer to the Measurement description in the zeta potential SOPs section of the main user manual for more details on each of the measurement options.
  • Page 56 Chapter 3 Surface zeta potential cell Settings Description The Tracer measurement displacement defines the displacement distance from the sample at which this FFR only (continued) measurement takes place. The displacement is altered in 125micron increments. Tracer measurement - Advanced Refer to the Measurement - Material description in the zeta potential SOPs section of the main user manual.
  • Page 57 Surface zeta potential cell Chapter 3 Settings Description Configure button Pressing the Configure button will display the Plate analysis parameters window, which enables various attributes of the analysis model to be altered. These include the measured zeta Display range, and the measurement thresholds.

  • Page 58: Performing The Measurement

    Chapter 3 Surface zeta potential cell Performing the measurement With the cell loaded into the instrument, and the SOP configured, a measurement can be performed. When the measurement is started, a user instruction is given to turn the cap on the top of the cell by a given amount - this will set the distance to the first required displacement position.
  • Page 59 Surface zeta potential cell Chapter 3 Once all of the specified measurements have been performed at that displacement, an instruction is given to set the cell to the next measurement position. This process will continue until all measurements have been made at all the positions specified in the SOP, and the surface zeta potential measurement is then complete.
  • Page 60 Chapter 3 Surface zeta potential cell A surface zeta potential report is available to view the results. Select View- Workspaces-Surface zeta potential to view the appropriate workspace. Editing the results Surface zeta potential results can be edited by right-clicking on the record in the records view and selecting Edit result;...

  • Page 61: Maintenance - Cleaning The Szp Cell

    Surface zeta potential cell Chapter 3 Maintenance - cleaning the SZP cell Caution! During cleaning and use it is vital not to let any fluid enter the top and cap area of the cell assembly. Any cross contamination of material from one measurement to the next could affect the result, so it is extremely important to ensure the cell is completely clean before use.

  • Page 62: Chemical Compatibility - Szp Cell

    Chapter 3 Surface zeta potential cell Intensive cleaning Cleaning can be performed as described in the following table. The material and chemical compatibility of each component is detailed in the next section. Component Cleaning method Cell cap Wipe clean with a mild soap solution. Rinse with water once cleaned.

  • Page 63: Flow-Mode Option

    Flow-mode option Introduction This chapter gives an overview of the Flow-mode option. Instruments fitted with this option can be connected to a size exclusion chromatography (SEC) system and be used as a light scattering detector. ZEN1006 Flow-mode option for Zetasizer Nano S and Zetasizer Nano ZS. It describes how to connect, control and operate the flow-mode arrangement to ensure reliable and consistent measurements.

  • Page 64: Applications

    Chapter 4 Flow-mode option In a flow-mode measurement, both the scattered light intensity and hydrodynamic diameter are plotted as a trend, and the addition of optional analogue inputs enables the simultaneous display of data from up to two other detector outputs. Liquid Connections Auto SEC/GPC...

  • Page 65: Connectivity

    Flow-mode option Chapter 4 Other detectors can be connected to the Zetasizer Nano optics unit and their signals plotted on the same axes as the light scattering signal. The timing of these signals can then be adjusted to compensate for the output delay due to the detectors being connected serially in the flow path.

  • Page 66: Exporting The Flow-Mode Data

    (additional) ill 7907 The lead supplied by Malvern can be connected directly to the rear of the external device, or to an output from the device, whichever is appropriate. Consult the external device documentation to obtain the output connections for connecting the Malvern lead.

  • Page 67: Editing And Inspecting A Flow-Mode Result

    Flow-mode option Chapter 4 Editing and inspecting a flow-mode result A flow-mode result can be edited by right-clicking on the measurement record and selecting Edit-Result. This will display the window below. ill 8822 The window shows the following:  Chromatogram This shows the flow-mode plot.
  • Page 68 Chapter 4 Flow-mode option  Traces The traces on the plot display the measured parameters with respect to the volume passed through the flow-mode system arrangement. Use the cursor to select each trace; the trace selected will be displayed in the plot legend and the displayed Trace tab.
  • Page 69 Flow-mode option Chapter 4  Viewing tools Scale (Control) (continued) Use to dynamically zoom in or out of the plot. This will be centred on the cursor position. The axes will automatically adjust to match the new scale. (Shift) Allows the plot to be moved left, right, up and down. The axes will automatically adjust to match the new position.
  • Page 70 Chapter 4 Flow-mode option  Measurement tab This Measurement tab displays the measurement details. These are the same details as entered when the measurement was first performed. Refer to the appropriate SOP windows - Sample and Flow settings - for more details. The Edit settings button will display the standard Edit result window where the measurement details and parameters can be inspected and edited.

  • Page 71: Controlling The Flow-Mode Measurement Via An Sop

    Flow-mode option Chapter 4 Controlling the flow-mode measurement via an SOP Once the Flow-mode option has been connected to the required size exclusion chromatography (SEC) system, a measurement can be made. This is done using an SOP, or a manual measurement in the usual manner. The Flow-mode SOP enables measurements to be performed on a flowing sample stream.
  • Page 72 Chapter 4 Flow-mode option Measurement type options Select a Flow measurement type then complete the SOP creation as required. External input 1 and 2 Measurement parameters can be set for each external input used. The same parameter options are available for each input. Settings Description With the Enable input check box selected, the input condi-...
  • Page 73 Flow-mode option Chapter 4 Settings Description Offset / Gain The Offset and Gain are mathematical parameters (values) that are needed to convert the incoming signal (in volts from the external measurement device) to the measurement parameter required. External Device Reading converted to voltage Parameter reading Signal input...
  • Page 74 Chapter 4 Flow-mode option Measurement Settings Description Measurement The measurement duration setting may affect the accuracy duration and repeatability of the results. In Measurement duration, input the total measurement time or volume amount required, and adjust the units to suit: Time or Volume.
  • Page 75 Flow-mode option Chapter 4 Flow settings Settings Description Flow rate Input the flow rate of the sample through the instrument and connecting tubing. This value is taken from the external detecting device (i.e. chromatographic column) that is used in the flow-mode measurement. For all other SOP windows, refer to the size SOP section.

  • Page 76: The Flow-Mode Measurement Display

    Chapter 4 Flow-mode option The Flow-mode measurement display The Flow-mode measurement displays are virtually identical to those shown when performing a standard size measurement. The only difference being the inclusion of different view options on the Result tab, i.e. Flow trace vs Volume as shown below.

  • Page 77: Displaying The Flow-Mode Report

    Flow-mode option Chapter 4 Displaying the flow-mode report To display a titration report, select a 'Flow' type measurement record and then select the appropriate report tab. The report will show all appropriate measurement information for that record. Standard report - flow-mode measurements 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.
  • Page 78 Chapter 4 Flow-mode option 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.

  • Page 79: Introduction

    Provides rheological parameters - G , η Data can be verified using the  same sample measured on a rotational rheometer where measurement ranges overlap. Data can be exported and used in the Malvern rSpace software. Zetasizer Nano accessories guide Page 5-1...
  • Page 80 Chapter 5 Microrheology Extends viscoelastic measurements into ranges inaccessible by mechanical  rheometry techniques. Fast measurements are possible, with all frequencies effectively sampled  simultaneously. Applications for viscosity of protein solutions and onset of protein aggregation.  DLS Microrheology - basic theory DLS Microrheology uses tracer probe particles to measure the relationship between stress and deformation.

  • Page 81: Microrheology Utilities

    Microrheology Chapter 5 Microrheology utilities The Zetasizer software has a utilities section for the Microrheology suite, which can be accessed via either the Tools menu (Tools-Utilities-Microrheology utilities) or from right clicking on a microrheology record. The following tab selections are available: Mean square displacement ...

  • Page 82: Exporting The Microrheology Data

    Chapter 5 Microrheology Exporting the Microrheology data On completion of the measurement the rheology data can be exported from the Zetasizer Nano software, saved as an .xml or a .csv file. Note The .csv file should be used if the results are to be imported into the Mal- vern rSpace software.
  • Page 83 Microrheology Chapter 5 On selecting OK, the data will be exported. The parameters that are exported  are: Sample Name Date File Name Lag times (μs) Times (μs) Angular Frequency (rad/s) Creep Compliance: Mean Square Displacement: Plot against Times Plot against Times Channel Values: Complex Viscosity (cP or mPas): The Correlogram, plot against Lag times...

  • Page 84: Controlling The Microrheology Measurement Via An Sop

    Chapter 5 Microrheology Controlling the Microrheology measurement via an SOP A microrheology measurement follows the same SOP format as performed when doing a normal size measurement, with a few exceptions. When a microrheology measurement is chosen some extra dialogues - Optimization and tracer - will be included in the SOP selections.
  • Page 85 Microrheology Chapter 5 depending upon the measurement setup and data available, both the zeta potential and size optimization measurements can be ignored, with the user going directly to the microrheology stage. Settings Description Zeta potential Select or deselect the zeta potential and size tick boxes as SOP / Size SOP required.
  • Page 86 Chapter 5 Microrheology Tracer The Tracer SOP window shows details about the tracer used in the measurement. Press Select to open the tracer manager window where the tracer to be used can be selected. Settings Description Select Highlight the tracer required and press OK. The Tracer SOP will now be populated with the selected tracer.

  • Page 87: Microrheology Measurement Process

    Microrheology Chapter 5 For a description of the other measurement parameters, refer to the Measurement - Advanced description in the size SOPs section of the main user manual. For all other microrheology SOP windows, refer to the size SOP  section in the main user manual.
  • Page 88 Chapter 5 Microrheology Sample preparation advice The correct preparation of the tracer and sample elements for the measurement is important to ensure that reliable and repeatable results are achieved. The concentration of tracer samples used will depend to a great extent on the concentration of your sample in the dispersant.
  • Page 89 Microrheology Chapter 5 The following starting concentrations are suggested. Tracer particles in measurement sample  Start with a few μl (up to about 5μl) of neat tracer to 1ml of sample. If more tracer is required to hide the sample scattering, add 1μl at a time. Alternatively;...
  • Page 90 Chapter 5 Microrheology For the microrheology measurement to measure reliably the acceptable zeta ratio is 25% or less. Note It is important to note that stable dispersion of the tracer particles in the sample may take some time. It has been noticed that with certain systems, gentle mixing (for example using a sample roller) will help to disperse the tracer particles, but that surface interactions can take several hours to man- ifest themselves.
  • Page 91 Microrheology Chapter 5 Zeta potential measurement to study sample/tracer interaction Measure tracer zeta potential in dispersant/buffer (or solvent) alone This is termed the tracer measurement. Use a zeta potential cell. A zeta potential cell is filled with the dispersant/buffer, that is to be used in the ...
  • Page 92 Chapter 5 Microrheology The tracer size measurement is used later in the microrheology measurement  step. The tracer size needs to be measured to ensure the microrheology result is as accurate as possible. When finished a dialogue will appear indicating the next step to perform. ...
  • Page 93 Microrheology Chapter 5 Size measurement to monitor tracer and sample scattering Measure size of measurement sample Use a folded capillary cell or disposable sizing cell Prepare a fresh measurement sample and perform a size measurement.  When finished a dialogue will appear indicating the next step to perform. ...
  • Page 94 Chapter 5 Microrheology With the measurement is complete, an intensity plot should also be displayed showing one peak. The software compares the two measured size results and tests for the following: The size of the tracer particle should be larger than the sample particles ...

  • Page 95: Displaying The Microrheology Measurement Report

    Microrheology Chapter 5 Running only the microrheology test The microrheology test can be run on it's own as long as the user is confident that the other steps have been fulfilled to their satisfaction. For example, if a range of concentrations of the same sample are being tested, the zeta potential testing can be run once, on just a single concentration.
  • Page 96 Chapter 5 Microrheology These are: Microrheology: Correlogram and MSD (mean squared displacement).  Rheological properties: Eta (Complex viscosity), G (Moduli), Creep  compliance. Microrheology MSD (mean squared displacement) The mean squared displacement is a representation of the movement of the tracer particles within the sample.
  • Page 97 Microrheology Chapter 5 A standard report (MSD) The standard microrheology report, MSD (mean squared displacement) (M), gives similar information as seen in a standard size and zeta potential report, plus additional information relating to the microrheology measurement itself. Zetasizer Nano accessories guide Page 5-19...
  • Page 98 Chapter 5 Microrheology Page 5-20 MAN 0487...

  • Page 99: Advanced Protein Features

    Advanced protein features Introduction This chapter gives an overview of the Advanced protein features option in the Zetasizer software. With the Zetasizer Nano ZSP it is now possible to achieve the best possible  measurement of protein mobilities. This is achieved with the combination of the following features: A system with sufficient sensitivity to measure the low count rates and low ...
  • Page 100 Chapter 6 Advanced protein features The basic steps involved in a measurement are: The first step is the thermal equilibration delay, performed in order for the  sample and cell to properly equilibrate with the Zetasizer cell holder. An optional pre-mobility size measurement is completed so that the user can ...
  • Page 101 Advanced protein features Chapter 6 Once all of the groups have been recorded the post-mobility size  measurement is performed in order to determine the state of the sample after the electrophoresis measurement. This completes the measurement process.  Diffusion barrier measurement technique Protein mobility measurements should be used in combination with the diffusion barrier measurement technique to further protect the sample.

  • Page 102: Controlling A Protein Mobility Measurement Via An Sop

    Chapter 6 Advanced protein features Controlling a protein mobility measurement via an SOP A protein mobility measurement is divided into two separate measurement parts - zeta potential and size. The protein mobility measurement follows the same SOP format as performed when doing a normal zeta potential or size measurement, with a few exceptions.
  • Page 103 Advanced protein features Chapter 6 Measurement Settings Description Sub-runs per With Automatic selected, the software will automatically group determine the numbert of sub-runs required per measure- ment. This will be suitable for the majority of samples and can simply be left as the default. With manual selected the measurement will use the user defined Number of runs: set- ting.
  • Page 104 Chapter 6 Advanced protein features Running a large number of sub-runs sequentially significantly increases the risk of Joule heating of the sample, so the protein mobility measurement is split into the smaller groups of sub-runs with a delay between groups to allow the sample to relax.

  • Page 105: Performing The Measurement

    Advanced protein features Chapter 6 Settings Description Partial results If it is likely that a measurement will not produce a correlation function that can be analysed, then the data collected can still be saved by selecting the Allow results to be saved containing correlation data only check box.

  • Page 106: Interpreting The Results

    Chapter 6 Advanced protein features Interpreting the results The record view As part of each group measurement, the count rate is measured and a rolling average taken as the measurement proceeds. Aggregates are, generally, characterised by a much larger particle size than the native protein and if any aggregates are present then large changes in the measured count rate in each group will be observed.
  • Page 107 Advanced protein features Chapter 6 Displaying the protein mobility measurement report To display a protein mobility report, select a Protein mobility group type measurement record and then select the appropriate report tab. The report will show all appropriate measurement information for that record. Standard report - protein mobility measurements Once a protein mobility measurement is completed there are a number of standard reports available for reviewing the measurement results.

  • Page 108: Calculators Tool

    Chapter 6 Advanced protein features Calculators tool One of the three basic functions of the Zetasizer Nano series of instruments is its ability to perform accurate measurement of a sample’s molecular weight. By measuring the sample scattering intensity over a range of concentrations and entering the necessary sample parameters, the molecular weight can be determined.
  • Page 109 A tool that estimates the ratio between monomer and dimer in a peak that contains both where the size of each is known. This is based on work published by Malvern Instruments entitled "Dynamic light scattering as a relative tool for assessing the molecular integrity and stability of monoclonal antibodies" by Nobbmann U et al.
  • Page 110 Chapter 6 Advanced protein features SLS Debye plot As mentioned in the introduction, the Calculators tool includes the ability to generate a Debye plot, using inputted rather than measured data. This feature can be useful for various reasons, for example: By combining individual measurements, one single Debye plot can be ...
  • Page 111 Advanced protein features Chapter 6 Note The Debye plot uses the reduced Rayleigh scattering equation. Adding & editing sample parameters & table data To generate an SLS Debye plot, the sample parameters and table concentrations have to be entered. To access the Debye plot select Tools-Calculators and then the SLS Debye plot tab.
  • Page 112 Chapter 6 Advanced protein features The table values and graph plot can be subsequently altered by changing the  Sample, Data and System parameters in the measurement parameters table on the right of the window. These parameters are described in the following section.
  • Page 113 Advanced protein features Chapter 6 The parameters are described below. Data This indicates the data entries that have been entered into the Debye plot table, see below. Sample Sample dn/dc (ml/g)  This is the specific refractive index increment; the change in refractive index as a function of the change in concentration.
  • Page 114 Chapter 6 Advanced protein features Copying the Debye plot The graph can be pasted into another application (such as Microsoft Word or Excel) by selecting the Copy button. Results area With both the table data and sample parameters entered the results will be automatically calculated and shown alongside the graph.
  • Page 115 Advanced protein features Chapter 6 Once the molecular weight - either measured or estimated - and the specific volume are known, the particle shape information can be estimated by using the Protein utilities tool. The Shape estimate calculator takes the entered data and then applies two equations - the Stokes-Einstein and the Perrin factor.
  • Page 116 Chapter 6 Advanced protein features : Hydrodynamic diameter. The diameter as measured via DLS. : The diameter by mass. This is calculated from the known molecular Mass weight and the specific volume of the particle. f : Particle frictional coefficient. ...
  • Page 117 Advanced protein features Chapter 6 To find the molecular weight estimate, enter the measured Hydrodynamic diameter value into the text box and the estimated molecular weight will automatically be calculated. The molecular weight is displayed in four ways: Globular Linear Branched poly- Starburst Proteins...
  • Page 118 Chapter 6 Advanced protein features Select the Concentration utilities tab to view concentration and scattering parameters. Concentration and Scattering This area of the window contains features to establish the concentration and scattering levels that may be observed from the sample. Enter the values from the measurement into the table.
  • Page 119 Advanced protein features Chapter 6 Enter the values from the measurement into the table and press Plot - the graph will update to show the values entered. To see the result of changing a value, change the required value and press Add plot - a new plot line will be added to the graph.
  • Page 120 Chapter 6 Advanced protein features = electrophoretic mobility Z = zeta potential ε = dielectric constant Ƞ = viscosity F(κa) = Henry's function. Henry's function (f(κa)) is defined as :  where: = dielectric constant ε = permittivity of free space (8.8542*10^-12 C/Vm) ε...
  • Page 121 Advanced protein features Chapter 6 and the other values can be taken from the above. Virial Diameter A tool to calculate the 'virial diameter' from the measured molecular weight and 2 virial coefficient (A2). Dynamic (DLS) Debye plot A dynamic Debye plot can be created by inputting sample parameters and data rather than using measured data.
  • Page 122 Chapter 6 Advanced protein features The format of the plot can be altered by choosing Chart properties from the drop-down menu in the top right hand corner of the graph. Adding and editing sample parameters and table data To access the Debye plot select Tools-Calculators and then the Debye plot tab. Select the Table tab to begin entering data into the table.
  • Page 123 Advanced protein features Chapter 6 Measurement parameters table When all the concentration values have been added into the table, use the measurement parameters table on the right hand side to alter the result and dynamic Debye plot. The parameters are: Molecular weight ...
  • Page 124 Chapter 6 Advanced protein features Interparticle distance This tool is used to calculate the distance between the particles based on their concentration and molecular weight. It also estimates the thickness of the electrostatic layer based on protein charge and ionic strength. The values are calculated according to the following formulae: Dielectric constant of the media ...
  • Page 126 Printed in England ill 8806 Malvern Instruments Limited Enigma Business Park Grovewood Road, Malvern Worcs, WR14 1XZ, U.K. Tel: +44 (0) 1684 892456 Fax: +44 (0) 1684 892789...

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