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Chapter 6

Advanced protein features

Page 6-20

MAN 0487

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. On entering each value,
press the return key afterwards and the results table will be updated.

Minimum Concentration Calculator

By entering only the molecular weight the Sample concentration (mg/ml) values
required for performing a measurement can be calculated:



Sizing sample concentration on a Zetasizer Nano

instrument.



Sizing sample concentration on a Zetasizer Nano

S90

instrument.



Zeta potential sample concentration on a Zetasizer Nano

instrument.

Scattering functions

A scattering function plot can be generated by inputting the measurement data in
the list on the right, in this window:

Summary of Contents for Zetasizer Nano

Page 1: ...Zetasizer Nano Accessories Guide MAN0487 Issue 2 0 June 2014...

Page 2: ...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...

Page 3: ...ncentration cell ZEN1010 2 25 Surface zeta potential cell ZEN1020 2 29 Surface zeta potential cell Introduction 3 1 Measurement technique 3 3 Preparation for measurement 3 4 Controlling an SZP measure...

Page 4: ...orheology Introduction 5 1 About DLS microrheology 5 2 Microrheology measurement process 5 3 Microrheology SOPs 5 4 Sample preparation advice 5 6 Running microrheology measurements 5 8 Analyzing micro...

Page 5: ...d the health and safety information in the basic guide before operating the system This manual focuses on specific issues of the Zetasizer Nano accessories that are not covered by the above manuals Ac...

Page 6: ...t ZEN1020 Surface zeta potential cell Cell intended for the measurement of the zeta potential at the sur face of a flat material in an aqueous environment Use with DTS0012 and PCS1115 cuvette describe...

Page 7: ...o fill each cell with sample and then insert into the Zetasizer Nano instrument Some accessories require configuration of the software and SOP parameters Where required the software controls are ident...

Page 8: ...s available for use with the Zetasizer Nano when connected to a SEC system It includes the flowcell ZEN0023 Connection use and operation of a flow mode optioned Zetasizer Nano is described in the Flow...

Page 9: ...for the Nano ZS The Zetasizer Nano ZSP has increased capability for the measurement of small and weakly scattering molecules and particles This increased sensitivity improves the measurement of zeta p...

Page 10: ...Chapter 1 Introduction and accessory range Page 1 6 MAN 0487...

Page 11: ...their use and application The aspects covered are 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 Na...

Page 12: ...e The two trays that hold the cuvettes can be removed for cleaning The cuvette holder provides a place to store the thermal cap during changeover of cells The cap is released by raising the cap and li...

Page 13: ...The optical quality of the cells is vitally important when performing molecular weight and low concentration protein measurements derived count rate 100kcps therefore glass or quartz type cuvettes sh...

Page 14: ...ty Good to very good Good to very good Minimum Sample volume 0 75ml 1ml Advantages Low cost Single use disposable no cleaning Use with MPT 2 Titrator No sample cross contamination Fast sample change o...

Page 15: ...0 C systems Zetasizer Nano S90 ZS90 Zetasizer V Requires careful filling to avoid bubbles Not resistant to organic solvents Unsuitable for use at high temperatures above 70 C Only applicable to system...

Page 16: ...tor Highest optical quality Can use nearly any dispersant Low sample volume Disadvantages Requires cleaning after measurement With manual use requires careful filling to avoid bubbles Requires cleanin...

Page 17: ...specific advice will be given Size cells and cuvettes All size cells should be rinsed cleaned with filtered dispersant before use Molecular weight cells and cuvettes All molecular weight cells should...

Page 18: ...e 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 t...

Page 19: ...sensitivity of the S90 and ZS90 means that they are unlikely to be suitable for the majority of molecular weight measurements Flowcells Flowcells will be filled during the measurement procedure Refer...

Page 20: ...n using the MPT 2 Titrator Always minimise the tubing within the cell area before inserting into the pinch valve channel Follow the instructions in the previous section on how to insert the cell Do no...

Page 21: ...area Cleaning cuvettes Two main types of cuvette are available Disposable polystyrene Do not clean and re use disposable cuvettes It gives inaccurate results Reusable glass or quartz The cleaning pro...

Page 22: ...provide a low cost alternative to previous reusable quartz capillary cells The stoppers can be replaced with Luer connectors to provide leak free connection to the optional MPT 2 Titrator Size measure...

Page 23: ...illing a cell for a protein mobility measurement involves a different technique Refer to the Advanced protein features chapter DTS1070 cell The cell name DTS1070 will be identified on the central sect...

Page 24: ...rt a cell stopper in each port Remove any liquid spilt on the electrodes contacts Note The stoppers must be fitted before a measurement is performed Ensure that one stopper is fitted firmly and the ot...

Page 25: ...t do form Turn the cell upright and continue to inject slowly until the sample is at the top of the electrodes Check again for bubbles in the cell Tap the cell gently to dislodge these ill 7938 Check...

Page 26: ...sation 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 illary cell The plates provide increased temperature stab...

Page 27: ...pillary cell orientation and insertion The clear folded capillary zeta potential cells can show significant differences in sample count rate depending on the orientation of the cell in the cell holder...

Page 28: ...below for connecting the cell for the Titrator Always minimise the tubing within the cell area before inserting into the pinch valve channel The tubing is attached to the folded capillary cell using...

Page 29: ...rodes The cell should then be flushed through with water as described below To clean the cell Fill one syringe with de ionised water or the dispersant Place the full syringe in one of the sample ports...

Page 30: ...er of samples can be prepared and the Dip cell inserted to measure each one in turn For aqueous samples the Dip cell can be used in conjunction with the disposable polystyrene DTS0012 For non aqueous...

Page 31: ...ween the sample electrodes The cuvette must not be filled more than the recommended maximum depth of 10mm Tilt the cuvette to a maximum angle of 45 Slowly insert the cell into the cuvette until the me...

Page 32: ...p cell and cuvette can be removed together If adequate purchase cannot be obtained on both parts then the following procedure is recommended Lift the Dip cell up out of the cuvette but before complete...

Page 33: ...ts especially between different types of sample Cross contamination between samples can seriously affect the results Caution Do not immerse the complete cell Only the sample electrodes must dip in to...

Page 34: ...ctrodes with pure dispersant 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 s...

Page 35: ...he electrodes Description ill 8450 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...

Page 36: ...e 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: ...8447 Remove the screw cap Separate the two halves of the cell by pulling the rear casing vertically away from the metal front Note how the electrode chambers and quartz measurement cell block are ass...

Page 38: ...minutes Rinse with water once cleaned Electrode chambers and port Electrode Chamber Scrub gently with interdental brush and Hellmanex then scrub with copious amounts of de ionised water Smaller inter...

Page 39: ...in this manual Description ill 8687 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...

Page 40: ...Chapter 2 General cells and cuvettes Page 2 30 MAN 0487...

Page 41: ...ype device and can be used with 1ml cell DTS0012 and PCS1115 It is incompatible with the MPT 2 Titrator The cell consists of a height adjustable sample barrel in which the sample is glued onto a sampl...

Page 42: ...components to prepare load and set the sample Surface zeta potential cell with palladium electrodes A 12 well plate for storing the samples 10 PEEK sample holders A screwdriver for cell tightening For...

Page 43: ...smotic surface flow while at distances further from the surface it will be dominated by the electrophoretic motion of the tracer itself The graph below shows a typical plot of reported zeta displaceme...

Page 44: ...in the next sections once these are com plete the measurement can be performed Loading the surface zeta potential cell with sample Inserting the surface zeta potential cell into the instrument Loadin...

Page 45: ...securely held in place for the duration of the experiment Inserting the surface zeta potential cell The insertion of the surface zeta potential cell into the Zetasizer Nano is done in three stages Fi...

Page 46: ...mbly 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...

Page 47: ...ing the tracer particles Sufficient suspension should be added to the cell so that the sample electrodes and screw are all completely submerged This is approximately 1 2 ml Fill the cuvette the prepar...

Page 48: ...e holder barrel and elec trodes are covered As the cell is inserted it displaces the sample so any bub bles will be pushed out from the top of the electrode gap Once the electrodes are covered bring t...

Page 49: ...ial cell from the drop down list Set the Attenuator to 11 To identify if your sample is aligned correctly open the instrument lid and rotate the cell cap clockwise in increments of approximately 1 8 t...

Page 50: ...e are described in the above manual The other SOP sections are specific to the SZP cell being used these are described below Also note that some of the other dialogue pages will alter slightly to acco...

Page 51: ...will be the only cell choice available The Zetasizer software will configure all settings and parameters to match this cell The default selection is the Surface zeta potential cell SZP measurement The...

Page 52: ...ements made at each displacement away from the surface and the length of any delay between repeat measurements SZP displacement The SZP displacement options define the distances to be used during a su...

Page 53: ...n options are the same as standard measurement duration options available during normal zeta potential measurements Tracer measurements The tracer measurements options define the number of repeat meas...

Page 54: ...o be set It is generally best to leave these set to default Size ranges and measurement thresholds can be applied to the analysis to filter spurious peaks prior to the analysis being performed These c...

Page 55: ...ow which enables various attributes of the analysis model to be altered These include the measured zeta Display range and the measurement thresholds If it is known that all particles within the sample...

Page 56: ...will set the distance to the first required displacement position This is a manual operation and the user must open the cell area lid turn the head of the cell the specified amount then close the lid...

Page 57: ...ven 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 measureme...

Page 58: ...m the main menu With the Edit result window open the Debye length model tracer material and dispersant properties can then all be changed The surface zeta potential edit result option allows points to...

Page 59: ...r the measurement it is recommended that this is disposed of and a new one used for all subsequent measurements General cleaning As a complete assembly the cell can be cleaned using de ionised water o...

Page 60: ...nst the materi als identified below before inserting a sample It is also recommended that a test is performed on the material with the sample before more permanent usage is undertaken With proper use...

Page 61: ...fitted an option part number label will be attached to the front of the cuvette holder The flow mode option is included as a standard fitment on the Zetasizer Nano ZSP Flow mode The Flow mode option...

Page 62: ...s to be monitored Applications Applications for this feature include use as a chromatography detector and a process monitor Separations detector The Zetasizer Nano can be connected to a size exclusion...

Page 63: ...and ZS means that many processes can be monitored without further sample preparation simplifying the measurement Note The method of sampling the process and transferring this to the Zetasizer is not p...

Page 64: ...ware saved as a text file and inserted into a spreadsheet software package such as Microsoft Excel for analysis This is done using the Export flow result macro option in the Tools menu If this macro i...

Page 65: ...will display the window below ill 8822 The window shows the following Chromatogram This shows the flow mode plot By default the Intensity trace is displayed on the left y axis and the Z average mean...

Page 66: ...e to select a region of the chromatogram which is highlighted in purple By right clicking the mouse it is then possible to perform a DLS analysis of this region This calculates and intensity weighted...

Page 67: ...ts dialogue Choose Con figure and then disable the option to Filter flow mode data to do this With reference to the originally attributed baseline level the software will display any peaks that may ha...

Page 68: ...plained later in this chapter Peaks tab The Peaks tab displays all the details about the calculated peaks shown in the flow mode plot To display a peak s details either select a blue peak bar in the p...

Page 69: ...put 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 type...

Page 70: ...With the Enable input check box selected the input condi tions required for the external signal can be setup Input name Use Input name to name the type of input Use a name that represents the signal o...

Page 71: ...external detector and the Zetasizer Nano This parameter ensures that data recorded by the Zetasizer can be over plotted from the same elution point This value will be obtained from the external detec...

Page 72: ...urement duration setting may affect the accuracy and repeatability of the results In Measurement duration input the total measurement time or volume amount required and adjust the units to suit Time o...

Page 73: ...all other SOP windows refer to the size SOP section Settings Description Flow rate Input the flow rate of the sample through the instrument and connecting tubing This value is taken from the external...

Page 74: ...he measurement progresses The result view will be updated after every run of the measurement The Result tab is named after the result view chosen the result view shown above is Flow trace vs Time Diff...

Page 75: ...ement 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 repor...

Page 76: ...e 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 displa...

Page 77: ...e microrheology option allows the measurement of the viscoelastic modulus of samples within the linear viscoelastic region This chapter provides the following information About DLS microrheology Micro...

Page 78: ...optimization protocol Advantages of DLS Microrheology Rh are Rheological characterization of low viscosity weakly structured samples Via access to very high frequency short time dynamics which is high...

Page 79: ...rocess consists of making three distinct measurements in this sequence 1 Tracer compatibility measurement zeta potential 2 Tracer concentration measurement size 3 Microrheology measurement Steps 1 and...

Page 80: ...solvent alone with no sample added sample is then added and a second measurement performed The two zeta potential results are then compared a small relative change in zeta potential in the presence o...

Page 81: ...particles this is usually the nominal diameter given on the bottle but ideally the hydrodynamic size of the tracer in the sample solvent should be used This can be determined from a standard size mea...

Page 82: ...l dispersant solvent 0 5 l ml Tracer particles in presence of sample add one drop of the dispersed diluted sample approx 0 05ml to approximately 5ml of the previously prepared tracer dispersion allowi...

Page 83: ...is due to concentration rather than interaction The tracer and sample are interacting If the tracer and sample are interacting adding more tracer will not improve the difference in zeta potential unl...

Page 84: ...he dispersant AND sample being used for the Microrheology test The test compares the two measured zeta potential results and tests to see if sample measurement is within a set tolerance of the tracer...

Page 85: ...the tracer particles 2 Start the measurement an initial measurement of the zeta potential of the tracer is performed and the following message is displayed 3 Click Next The tracer particle zeta poten...

Page 86: ...the tracer particles relative to that of the sample Using the relative scattering ratio and concentration entered the software will then calculate the concentration of tracer particles required to gi...

Page 87: ...l parameters are calculated The process measures the correlation function of the probe tracer particles in the sample matrix under test The Mean Square Displacement MSD of probe particles is then calc...

Page 88: ...the sample solvent This value can be modified by ticking the Edit box It is this value which is used in the microrheology calculation An optional smoothing window can be applied to the Mean Square Dis...

Page 89: ...Space Microrheology reports There are a number of standard reports available for reviewing the measurement results Microrheology Correlogram and Mean Squared Displacement MSD Rheological properties Co...

Page 90: ...osity and shear viscosity increases as the sample structure becomes more complex Viscoelastic Modulus G The Moduli tab shows the viscoelasticity of the sample it shows two sets of data on the chart G...

Page 91: ...o the microrheology data It is important to note however that not all models will be applicable to all the datasets Due to the measurement inconsistencies at very short timescales there will be areas...

Page 92: ...stination Alter the file extension to xml file or csv as required 4 Click OK to export the data The parameters exported are Note This data can be imported into rSpace software using an appropriate seq...

Page 93: ...iffusion barrier measurement technique a description of this follows A measurement process that reduces the risk of aggregation but also capable of identifying any aggregates that are formed and asses...

Page 94: ...all recorded sub runs is reported into the record view During the measurement proper running a large number of sub runs sequentially will significantly increase the risk of Joule heating of the sample...

Page 95: ...lectrodes The diffusion barrier technique protects the protein from damage by introducing a physical distance between the sample and the electrodes This is done by holding a plug of measurement sample...

Page 96: ...ein mobility measurement being performed these are described below Also note that some of the other dialogue pages will alter slightly to accommodate extra parameters necessary to perform the microrhe...

Page 97: ...epeatability of the measurement of particularly polydisperse samples All the individual runs are accumulated and then averaged to give a final zeta potential result Therefore the more runs performed t...

Page 98: ...tion Perform a size measurement With the Perform a size measurement before and after the Protein mobility measurement check box selected a size measurement will also be performed as well as the standa...

Page 99: ...mobility measurement displays are identical to those shown when performing a standard zeta potential measurement The measurement and a a description of the measurement tabs is explained in the Zetasi...

Page 100: ...once aggregated groups have been removed The average over un aggregated groups will yield the mobility of only the protein itself and not its aggregates but the number of groups included in the measu...

Page 101: ...surement 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 m...

Page 102: ...ollowing tab selections are available SLS Debye plot The protein utilities function enables a static light scattering Debye plot to be constructed from freely available information or from a record ge...

Page 103: ...tion and molecular weight Also estimates the thickness of the electrostatic layer based on protein charge and ionic strength Mixture Viscosity A simple tool that calculates the overall viscosity of a...

Page 104: ...s Any of the parameters in the window box can be changed the other parameters will be instantly recalculated This can be used to investigate the sensitivity of the result to changes in any parameter F...

Page 105: ...e Table tab to begin entering data into the table To define a new concentration press the Add button The table Input values window will be displayed Specify the Concentration and Sample intensity valu...

Page 106: ...s are described in the following section Measurement parameters table Once all the concentration values have been added into the table the measurement parameters table can be used to alter the result...

Page 107: ...the residual count rate which is derived by subtracting the solvent count rate from the sample count rate Shape Model The shape model that is used to estimate the radius of gyration from the hydrodyna...

Page 108: ...ength between the molecule and the solvent This is calculated from the slope of the plot K Mol cm2 g The instrument optical constant Molecular weight MW and shape estimates The hydrodynamic size measu...

Page 109: ...sion coefficient D now known the Hydrodynamic diameter can be calculated by using a variation of the Stokes Einstein equation DH Hydrodynamic diameter k Boltzmann constant f Particle frictional coeffi...

Page 110: ...r F plus the Prolate and Oblate axial ratio will be automatically calculated and displayed in the results area If required a solvent layer can be subtracted from the hydrodynamic radius when calculati...

Page 111: ...nd the estimated molecular weight will automatically be calculated The molecular weight is displayed in four ways Hydrodynamic diameter estimate This works in the same way as above except the molecula...

Page 112: ...value press the return key afterwards and the results table will be updated Minimum Concentration Calculator By entering only the molecular weight the Sample concentration mg ml values required for pe...

Page 113: ...Protein charge Ka This tool is used to calculate Ka for the Henry equation using known size and ionic strength This tool uses the Ohshima equation for monovalent salts The second calculation calculate...

Page 114: ...gadros number 6 022 10 23 mole K Boltzmann s constant 1 38065 10 23 m2kg s2K T temperature K I ionic strength moles L This calculator allows a more specific value other than the Smoluchowski or Huckel...

Page 115: ...A dynamic Debye plot can be created by inputting the concentration points from an existing measurement then additional concentration points can be added Any of the parameters in the dialogue box can...

Page 116: ...ed Specify the Concentration and Measured diffusion coefficient values either new values or ones taken from an existing measurement To modify a concentration select it from the list and press the Modi...

Page 117: ...is completed the other will be updated Saving the dynamic Debye plot Click Save to save the parameters and other inputted data To reviewed the saved parameters at a later stage click the Load button...

Page 118: ...ere o permittivity of free space 8 8542 10 12 C Vm inverse Debye length 1 nm NA Avogadros number 6 022 10 23 mole K Boltzmann s constant 1 38065 10 23 m2kg s2K T temperature K I ionic strength moles L...

Page 119: ...2 7 2 13 Flow settings SOP 4 13 Flowcell 2 10 Flow mode 4 1 4 9 Connectivity 4 3 Exporting the data 4 4 Process monitor 4 3 Flow mode Editing 4 5 Flow mode Inspecting 4 5 Flow mode measurements 4 14...

Page 120: ...9 Result tab Flow mode 4 14 S Scattering 6 20 Scattering functions 6 10 Second virial coefficient 6 16 Serial number 2 2 Shape 6 10 Shape estimate calculation 6 18 Shape model 6 15 Size measurement S...

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