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Malvern Instruments Zetasizer Nano Accessories Manual

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Zetasizer Nano
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Guide
MAN0487 Issue 2.0 June 2014

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Summary of Contents for Malvern Instruments Zetasizer Nano

  • Page 1 Zetasizer Nano Accessories Guide MAN0487 Issue 2.0 June 2014...
  • Page 2 Malvern Instruments shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance or use of this material.

  • Page 3: Table Of Contents

    ......4-4 Exporting the flow-mode data ....4-5 Editing and inspecting a flow-mode result Zetasizer Nano accessories guide Page i...
  • Page 4 Table of Contents Zetasizer Nano accessories guide ... . 4-9 Controlling the flow-mode measurement via an SOP ..... 4-14 The Flow-mode measurement display .

  • Page 5: Introduction And Accessory Range Introduction

    Accessory range The accessories available for use with the Zetasizer Nano are indicted in the fol- lowing section. Full descriptions on usage and application are contained in the sub- sequent chapters. The accessories that can be used will be dependent upon the instrument configuration and measurement type that will be performed.
  • Page 6 Size and molecular weight measurements DTS1070 Folded capillary cell - This cell can be used for size measure- ments, in the Zetasizer Nano S, Nano ZS and Nano ZSP only. (This cell is a direct replacement for DTS1060/61). DTS0012 Square polystyrene cuvettes - for size and molecular weight meas- urements.
  • Page 7 Identification of each cell of the dispersion units with guidance on selection.  How to fill each cell with sample and then insert into the Zetasizer Nano  instrument. Some accessories require configuration of the software and SOP parameters.
  • Page 8 Instruments fitted with the flow-mode option can be connected to a size exclusion chromatography (SEC) system and be used as a light scattering detector. The following kit is available for use with the Zetasizer Nano when connected to a SEC system. It includes the flowcell ZEN0023...
  • Page 9 For use with all Zetasizer Nano instruments.  Microrheology Microrheology is a new measurement type available to users of the Zetasizer Nano ZS and ZSP. It allows the measurement of the viscoelastic modulus of samples within the linear viscoelastic region.
  • Page 10 Chapter 1 Introduction and accessory range Page 1-6 MAN 0487...

  • Page 11: General Cells And Cuvettes

    Identification of each cell of the dispersion units with guidance on selection.  How to fill each cell with sample and then insert into the Zetasizer Nano  instrument. Some accessories require configuration of the software and SOP parameters.

  • Page 12: 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 13: Cell And Cuvettes

    (derived count rate <100kcps), therefore glass or quartz type cuvettes should be used to ensure the optimum signal is achieved. Briefly the following cells can be used with the Zetasizer Nano instrument. Cell Application Disposable “polystyrene”...
  • Page 14 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 15 Unsuitable for use at high Unsuitable for use at high temperatures. (above 70°C) temperatures. (above 70°C) Only 90°C systems Only applicable to systems (Zetasizer Nano S90, ZS90, with NIBS optics Zetasizer μV) (Zetasizer Nano S/ZS/ZSP) Material Polystyrene Polystyrene Glass - square aperture...
  • Page 16 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 17 If using syringe filters for the dispersant, never use the first few drops from the  syringe, in case there are any residual dust particles in the filter that may con- taminate the dispersant. Zetasizer Nano accessories guide Page 2-7...

  • Page 18: 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 19 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 for the Zetasizer Nano S, ZS and ZSP, 2 microlitres for the Zetasizer V and 20 microlitres for the Zetasizer μ...
  • Page 20 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 21 Try submerging the cuvette in an ultrasonic bath of clean solvent.  Once clean, wipe the cuvette with a lint free tissue (photographers’ lens clean-  ing tissues are recommended). The smaller and more dilute the sample, the more important cleanliness is.  Zetasizer Nano accessories guide Page 2-11...

  • Page 22: Folded Capillary Cell (Dts1070 / Dts1060/61)

    Chapter 2 General cells and cuvettes Folded capillary cell (DTS1070 / DTS1060/61) Description These are maintenance-free capillary cells primarily designed for zeta potential measurements, but can also be used for size measurements. They have been designed to be used for a single measurement or series of measurements, then discarded rather than cleaned.
  • Page 23 Slowly inject the sample from its syringe into the cell, filling the U tube to just  over half way . Check no air bubbles form in the cell. Tap the cell gently to dislodge any that  do form. ill 7938 Zetasizer Nano accessories guide Page 2-13...
  • Page 24 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 25 Check again for bubbles in the cell. Tap the cell gently to dislodge these.  ill 7938 Check that the electrodes are completely immersed.  Remove the syringe and insert a cell stopper in each port.  Remove any liquid spilt on the electrode contacts.  Zetasizer Nano accessories guide Page 2-15...
  • Page 26 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 27 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 28 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 29 Repeat the flushing process five more times, flushing the liquid backwards and  forwards between the syringes. The cell is then ready for use. Never attempt to clean the outside of the folded capillary cell. It causes small surface scratches that give inaccurate results. Zetasizer Nano accessories guide Page 2-19...

  • Page 30: Dip Cell (Zen1002)

    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 31 - a ‘stop’ on the Dip cell must rest on the top of the cell holder. Check that the cell sits flat on the cell holder. Zetasizer Nano accessories guide Page 2-21...
  • Page 32 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 33 Use the dispersant used for the previous sample as the cleaning fluid. If this dispersant contains additives such as surfactants, follow this by ultrasonicating for two minutes in the pure solvent. Zetasizer Nano accessories guide Page 2-23...
  • Page 34 Chapter 2 General cells and cuvettes ill 6763 Warning! Take care: ultrasonication can produce a fine aerosol of the bath liquid. Remove the electrodes from the bath and rinse them with pure solvent. A pipe  cleaner can be used for gentle cleaning of electrodes. To protect the Dip cell after cleaning, we recommend placing it in an empty ...

  • Page 35: High Concentration Cell (Zen1010)

    Check no air bubbles form in the cell. Tap the cell gently to dislodge any that  do form. Remove the syringe and insert a cell stopper in each port.  Remove any liquid spilt on the electrodes.  Zetasizer Nano accessories guide Page 2-25...
  • Page 36 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 37 Detach the pipework and remove the top port .  Protect the cell block from damage .  Once the cell has been disassembled, cleaning can be performed as described in the following table. Zetasizer Nano accessories guide Page 2-27...
  • Page 38 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 39: Surface Zeta Potential Cell (Zen1020)

    MPT-2 Titrator. ill 8687 Application The surface zeta potential cell is used for measurements of aqueous samples. For details for this cell, refer to the separate Surface zeta potential chapter in this manual. Zetasizer Nano accessories guide Page 2-29...
  • Page 40 Chapter 2 General cells and cuvettes Page 2-30 MAN 0487...

  • Page 41: 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 42 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 43: Measurement Technique

    In the displacement graph above, the blue circles represent the reported zeta potential of the tracer particles, while the red squares represent the zeta potential of the tracer particles measured far from the sample surface and also independent from any electro-osmotic effects. Zetasizer Nano accessories guide Page 3-3...

  • Page 44: 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 45 Inserting the surface zeta potential cell The insertion of the surface zeta potential cell into the Zetasizer Nano is done in three stages. First a coarse alignment is performed where the cell has to be aligned to a zero ...
  • Page 46 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 47 This is to ensure the orientation is correct when inserted into the cell holder. Zetasizer Nano accessories guide Page 3-7...
  • Page 48 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 49 Any movement of the cell will result in a dif- ferent zero height and this must be constant throughout a given measure- ment. Any alteration of the height during an experiment will reduce the quality of the data. Zetasizer Nano accessories guide Page 3-9...

  • Page 50: Controlling An Szp Measurement Via An Sop

    Tracer measurement - will be included in the SOP selections. The SOP Editor and setup is described in full in the Zetasizer Nano user manual. Most of the SOP sections are common to Measurement types, and these are described in the above manual. The other SOP sections are specific to the SZP cell being used;...
  • Page 51 The SZP 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. Zetasizer Nano accessories guide Page 3-11...
  • Page 52 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 53 (FFR) only measurement. The purpose of this is to make a measurement only of the tracer mobility, which will not include any electro-osmotic component, and this will be used in the surface zeta potential equation. Zetasizer Nano accessories guide Page 3-13...
  • Page 54 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 55 Display range can be set to improve the repeatability of the measurement result; similarly a lower threshold sets the noise rejection baseline in the zeta potential distribution. Refer to the Help file for more information. Zetasizer Nano accessories guide Page 3-15...

  • Page 56: 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 57 SOP, and the surface zeta potential measurement is then complete. The data is stored as a ‘parent’ surface zeta potential record, with ‘child’ records relating to the individual zeta potential measurements made at each displacement. Zetasizer Nano accessories guide Page 3-17...
  • Page 58 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 59: Maintenance - Cleaning The Szp Cell

    6763 Over time, it is likely that the electrodes will become discoloured or tarnished. This is expected, and although it cannot be cleaned, this will not affect the quality of the data obtained. Zetasizer Nano accessories guide Page 3-19...

  • Page 60: Chemical Compatibility - Szp Cell

    Chemical compatibility - SZP cell Components of the Zetasizer Nano that may come into contact with the sample are manufactured from materials that are considered to give the widest protection from chemical attack. However, it is important to check that any sample or titrant used is chemically compatible with the materials mentioned.

  • Page 61: Flow-Mode Option

    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. The following cell is available for use with the Zetasizer Nano when connected to a SEC system. ZEN0116 Quartz flow cell kit for connection of the Zetasizer Nano to an SEC system.

  • Page 62: Applications

    By connecting the output from the external device to the external input socket on the rear of the Zetasizer Nano, a real-time parameter reading from the device can be directly inputted back into the Zetasizer Nano software. This parameter reading can be plotted as a trend, thus enabling additional sample characteristics to be monitored.

  • Page 63: Connectivity

    7906 Process monitor The Zetasizer Nano can be connected to a flowing sample extracted from a process stream or reaction vessel. The high concentration capability of the Nano S and ZS means that many processes can be monitored without further sample preparation, simplifying the measurement.

  • Page 64: Exporting The Flow-Mode Data

    Exporting the flow-mode data Once received, the external device data obtained during the flow-mode measurement can be exported from the Zetasizer Nano software, saved as a text file, and inserted into a spreadsheet software package (such as Microsoft Excel) for analysis.

  • Page 65: Editing And Inspecting A Flow-Mode Result

    Z-average mean parameter displayed on the right y-axis. Right- clicking on the y-axis text allows other available traces to be displayed. The available traces are displayed in the Selected trace area of the Traces tab. Zetasizer Nano accessories guide Page 4-5...
  • Page 66 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 67 Trace data. Options will be given to either remove the highlighted section (Delete data in this section only), or to remove all data out- side the highlighted section (Keep data in this section only). Zetasizer Nano accessories guide Page 4-7...
  • Page 68 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 69: Controlling The Flow-Mode Measurement Via An Sop

    - External Input 1/2 and Flow settings - will be included in the SOP selections. The SOP Editor and setup is described in full in the Zetasizer Nano user manual. Most of the SOP sections are common to Measurement types, and these are described in the above manual.
  • Page 70 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 71 The Delay volume is the volume of liquid contained in the cells and connecting tubing between the external detector and the Zetasizer Nano. This parameter ensures that data recorded by the Zetasizer can be over-plotted from the same elution point.
  • Page 72 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 73 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. Zetasizer Nano accessories guide Page 4-13...

  • Page 74: The Flow-Mode Measurement Display

    Result tab, i.e. Flow trace vs Volume as shown below. The standard tabs are explained in the Zetasizer Nano manual. 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.

  • Page 75: Displaying The Flow-Mode Report

    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. Zetasizer Nano accessories guide Page 4-15...
  • Page 76 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 77: Introduction

    Introduction This chapter gives an overview of the microrheology option in the Zetasizer software. It can be used with either a Zetasizer Nano ZS or ZSP. The microrheology option allows the measurement of the viscoelastic modulus of samples within the linear viscoelastic region.

  • Page 78: About Dls Microrheology

    Chapter 5 Microrheology About DLS microrheology The term for the measurement protocol that a Microrheology optioned Zetasizer Nano instrument performs is DLS Microrheology (μRh). This technique uses dynamic light scattering to determine the rheological properties of low viscosity and weakly structured samples by measuring the motion of an embedded tracer particle.

  • Page 79: Microrheology Measurement Process

    These stages are not always necessary and depend upon the measurement setup and data available. It is possible to proceed directly to the microrheology measurement (3) if required. The optimization stages are shown in the flow charts below: Zetasizer Nano accessories guide Page 5-3...

  • Page 80: Microrheology Sops

    Chapter 5 Microrheology Microrheology SOPs Creating a microrheology SOP or manual measurement is very similar to that of a standard SOP, except that one of the Microrheology measurement types is selected: Measurement type Description Microrheology Zeta Potential measurement to check there is no optimization –...
  • Page 81 Useful when measuring the forward angle. Microrheology The (odd) number of points in the smoothing smoothing window used to smooth the window size MSD (Mean Squared Displacement) data. The available settings are: No smoothing, 3, 5, 7, 9. Zetasizer Nano accessories guide Page 5-5...

  • Page 82: Sample Preparation Advice

    Chapter 5 Microrheology Sample preparation advice Correct preparation of the tracer and sample elements is important in ensuring the reliability and repeatability of the results. The concentration of tracer samples depends to a great extent on the concentration of your sample in the dispersant. The optimization steps for Tracer Compatibility and Tracer Concentration are useful tools for assessing whether the tracer chemistry is likely to be suitable for a particular sample, and if so the minimum tracer concentration that can be used.
  • Page 83 Tracer Concentration field in the SOP’s Tracer properties section. Filtering  It is suggested that to get the most reproducible result, the tracers are filtered with appropriately sized (i.e. filter size larger than the tracers) filter. Zetasizer Nano accessories guide Page 5-7...

  • Page 84: Running Microrheology Measurements

    Chapter 5 Microrheology Running microrheology measurements Use either a manual measurement or SOP to run one of the microrheology measurement types. Tracer compatibility measurement (zeta potential)  Tracer concentration measurement (size)  Microrheology measurement  During the measurement’s progress the display is similar to that of a standard zeta potential or size measurement, with the addition of extra information pop-up windows.
  • Page 85 Click Next. The tracer particle zeta potential measurement is made and the result shown: Follow the instructions given and click Next. The variance from the Acceptable Zeta Difference is displayed: Zetasizer Nano accessories guide Page 5-9...
  • Page 86 Chapter 5 Microrheology If the difference is less than 5mV then the measurement has been suc-  cessful - proceed to make a Tracer concentration measurement. If the difference is more than 5mV the software will provide sample prep-  aration advice and advise on repeating the measurement.
  • Page 87 This step is not included as an optimization step in the microrheology application as it can be determined from a standard size measurement of the tracer in the solvent. Zetasizer Nano accessories guide Page 5-11...
  • Page 88 Chapter 5 Microrheology Some tracer particles are already set-up in the Sample-Tracer window under Tracer Details. These include tracer particles supplied by Malvern and can be 'edited' or 'added to' by the user as required. For Malvern supplied particles the Nominal Tracer Size is usually the size on the bottle which may differ from the hydrodynamic size in the sample solvent.

  • Page 89: Analyzing Microrheology Data

    To display a microrheology report, select a Microrheology measurement record and then select the appropriate report tab. The report will show all appropriate measurement information for that record. This is an example of the Mean Squared Displacement Report (MSD): Zetasizer Nano accessories guide Page 5-13...
  • Page 90 Chapter 5 Microrheology Mean Squared Displacement (MSD): a representation of the movement of the tracer particles within the sample. A purely viscous (Newtonian) sample will show a straight line. Correlogram: shows the base data from which the microrheology result is calculated.
  • Page 91 To export the microrheology data, refer to the Microrheology data topic. Exporting microrheology data Following the completion of the measurement, the rheology data can be exported from the Zetasizer Nano software as an .xml or a .csv file. Zetasizer Nano accessories guide Page 5-15...
  • Page 92 Chapter 5 Microrheology Note The .csv format should be used if the results are to be imported into the Malvern rSpace software. Exporting the microrheology data (.xml/.csv) - using the Microrheology utilities: Select a microrheology measurement record. Select Tools-Utilities- Microrheology utilities. The Microrheology data processing window is displayed.

  • Page 93: Advanced Protein Features

    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...
  • Page 94 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 95 The sample sits at the bottom of the cell if the cell is handled carefully. Advantages of this technique are that: Many more measurements can therefore be made before aggregates start to  appear. Only small volumes (20-50 μl) are required.  Zetasizer Nano accessories guide Page 6-3...

  • Page 96: Controlling A Protein Mobility Measurement Via An Sop

    SOP format as performed when doing a normal zeta potential or size measurement, with a few exceptions. The SOP editor and setup is described in full in the Zetasizer Nano user manual. Most of the SOP sections are common to Measurement types, and these are described in the above manual.
  • Page 97 Add a delay between the measurements of each group in the Delay between groups entry box if required. A typical delay between groups would be 180s. Zetasizer Nano accessories guide Page 6-5...
  • Page 98 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 99: Performing The Measurement

    The measurement and a a description of the measurement tabs is explained in the Zetasizer Nano user manual. During the measurement the data is stored as a ‘parent’ protein mobility zeta potential record, with ‘child’ records relating to the individual zeta potential and size measurements made during each measurement group.

  • Page 100: 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 101 The main reports are Protein mobility and Protein analysis. An example of the Protein mobility report is shown above. The report gives similar information as seen in a standard size and zeta potential report, plus additional information relating to the protein mobility measurement itself. Zetasizer Nano accessories guide Page 6-9...

  • Page 102: 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 103 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 104 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 105 The Input values window is displayed, allowing the parameters to be changed. A concentration can be deleted by selecting the concentration from the list  and pressing the Delete... button. Select the Graph tab to see the resultant Debye plot.  Zetasizer Nano accessories guide Page 6-13...
  • Page 106 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 107 KC/RoP for particles of sizes outside the Rayleigh region (Diameter > ~50nm). System Wavelength (nm)  The wavelength of the laser used in the Zetasizer Nano instruments, or for the measurement. Either 632.8nm ‘red’ or 532nm ‘green’ laser wavelengths are available. Scattering angle (degrees) ...
  • Page 108 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 109 6D The Perrin factor (F) is : ---------- - ------------------------ - ------------- - ------------- - 6D Mass Mass Mass Mass Zetasizer Nano accessories guide Page 6-17...
  • Page 110 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 111 Branched poly- Starburst Proteins polysaccharides saccharides polymers Hydrodynamic diameter estimate This works in the same way as above, except the molecular weight (in kDaltons) is entered to find the Hydrodynamic diameter instead. Concentration utilities Zetasizer Nano accessories guide Page 6-19...
  • Page 112 Sizing sample concentration on a Zetasizer Nano S instrument.  Sizing sample concentration on a Zetasizer Nano S90 instrument.  Zeta potential sample concentration on a Zetasizer Nano Z instrument.  Scattering functions A scattering function plot can be generated by inputting the measurement data in...
  • Page 113 Stokes radius. Henry equation The basic zeta potential measurement performed by the Zetasizer Nano measured the electrophoretic mobility of the particle or molecule under investigation. The mobility is related to the zeta potential using the Henry equation where:...
  • Page 114 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 115 - e.g. sample temperature - can be altered, with the effect immediately observed on the Debye plot. This saves time in performing the original measurement again at the different temperature. Zetasizer Nano accessories guide Page 6-23...
  • Page 116 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 117 DLS interaction parameter  The DLS interaction parameter (kD) is also known as the dynamic virial coefficient and is related to the slope of the line on this plot. Frictional coefficient  The frictional coefficient. Zetasizer Nano accessories guide Page 6-25...
  • Page 118 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 ε...
  • Page 119 Low volume Glass flow cuvette External Input 1 and 2 (SOP) Low volume quartz cuvette 4-10 Filling the cell 2-7, 2-13 Measurement (SOP) 4-12 Flow settings (SOP) 4-13 Measurement tab Flowcell 2-10 Microrheology Flow-mode 4-1, 4-9 Zetasizer Nano accessories guide Page i...
  • Page 120 Index Zetasizer Nano accessories guide Minimum Concentration Calculator Serial number 6-20 Model number Shape 6-10 Molecular weight Shape estimate calculation 6-18 Calculations Shape model 6-16 6-15 Estimate Size measurement (SOP) 6-18 Molecular weight calculations SLS Debye plot 6-10 6-10 MPT-2 Autotitrator...

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