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User Manual For The DynaPro Molecular Sizing Instrument And

1. UserDefined Cauchy Coef nm Measurements il Temp Model Meas 1 a Meas 2 Meas 3 Click here to select k the measurement Aqueous User Defined User Defined Measurement Meas1 DYNAMICS V6 is delivered with an integrated Solvent Database containing roughly 100 predefined solvents To select a solvent click the list box for the Name property and select a solvent from the options contained within For those wishing to define their own solvent parameters a User Defined option is included in the Name list box When the User Defined option is selected all the solvent properties can be edited directly in the value cell A brief description of each solvent property follows Name This is the name assigned to the solvent All solvent property settings are linked to the solvent name selected from the list box in the value cell Parameter Source This property is used to define the source of the solvent property settings for a given solvent Solvents built from the database of known additives are designated as Standard All others are designated as User Defined Rfr Idx 589nm amp 20C This is the refractive index of the solvent at a wavelength of 589 nm and a temperature of 20 C Viscosity 20C This is the viscosity in cpoise of the solvent at a temperature of 20 C PROTEINSOLUTIONS www protein solutions com 78 Cauchy Coefficient The Ca
2. a QuickStart pst Files of type PreSet Experiments pst Cancel Z To open communications with the DynaPro select the Hardware Node in the Tree View and click the Connect button below the properties table Once communications are established the status will be reported in the Connection Status text box and the Record button filled circle icon on the tool bar will turn green indicating that the software is ready to begin recording data PROTEINSOLUTIONS www protein solutions com 40 DYNAMICS V6 Exp1 of Xx Parameters Measurements Optics Serial Number Analog Input 1 Analog Input 2 Analog Input 3 A Serial COM 1 Tree View i Serial COM 2 Connection Status Ku a e e 7 Disconnect Connect to the DynaPro by clicking here Measuring the Microsphere standard For Help press F1 The begin collecting data for the Microsphere standard you should first insure that the appropriate experimental parameters are loaded and that the instrument is connected When DYNAMICS V6 is first installed the default experimental settings are those suggested for measuring the Microsphere standard If you suspect that the default settings have been changed you can open a new Experiment Window with the Microsphere experimental parameters pre loaded by first selecting the File Open PreSets option on the main menu bar and then selecting the QuickStart PreSet in the PreSets folder
3. 50 Exp3 B2 Em Dla El Exp3 Hardware Property Value Parameters Fixed Instrument Solvent Sample 1 Select the parameter g to be added here 3 Enter the value of easements the parameter here Measurement Next Parameter 2 Use the Add button to add the parameter to the table Parameter lonic strength M x Remove It should be recognized here that DYANAMICS V6 does use any of the parameters in the User Defined sub node for calculation purposes If a parameter is required for a calculation such as the protein extinction coefficient for concentration calculations the parameter will be listed in one of the other Parameter sub nodes Notes amp Tips 1 If you routinely use a particular set of parameters the File Save Settings command on the main menu bar can be used to save the parameters and their values if entered such that they are always loaded when a new experiment is started Collecting data To begin collecting data for a sample you should first insure that the appropriate experimental parameters are loaded and that the instrument is connected To connect the instrument select the Hardware Node in the Tree View and click the Connect button below the properties table Once the instrument is connected the Record button on the Experiment Window tool bar will turn green indicating that the software is
4. Export This command exports selected data in text format to a user defined file Data Filter The Data Filter command opens a special window that allows the user to design automated self marking routines Table Settings This right click menu command opens a design window for table formatting The Table Settings window allows the user to add remove and order columns and rows set default units and set the font size Statistics The Statistics command opens or closes the Statistics Table at the bottom of the data table in the Grid View Column arrangement and formats in the Statistics Table are identical to those in the main data table The types of statistics displayed are user definable in the Table Settings window PROTEINSOLUTIONS www protein solutions com 83 fy File Edit Vier Right click here to remove a column D W or format the data in the column oe E MSX qa3 exp Right click here to open the Units Menu Hardware Intensity Baseline E Parameters E Measurements lngstroms 3 Acq 1 umJeters Acq2 H cm eters 7 2116710 2122260 2109050 2098470 2120440 2124220 1 2108640 49 i at a Select this option to open the Format Data window General Scientific Decimals jo Alignment F Left Center Right
5. Property Original Equipment AD Unit Hamilton pH probe 7 Host Units Model amp Serial Number Hamilton pH probe Optics Blocks Detector Type pH AMD Detectors Auxiliary Devices Auxiliary device properties Property Value Auxiliary Device Test Pump Original Equipment C Host Units Model amp Serial Number Test Pump K7 Optics Blocks Aux Device Type HPLC Pump C A D Detectors Kuonesooreonean i roonoroonsosoesaonons Notes amp Tips 1 Only 99E host units include a chip containing the original equipment properties 2 The DynaPro will only support A D detectors that generate positive signals between 0 and 2000 mV Negative potential signals can damage the internal circuitry Please contact your local Protein Solutions sales representative if you need an A D bias adapter suitable for use with your detector 3 Analog detectors are calibrated at the appropriate hardware sub node in the Experiment Window 4 You can define a new unit with the same properties as an existing unit by selecting the existing unit in the Host Optics Unit list box and then editing the Serial Number Defining the hardware configuration The hardware configuration for a new experiment is defined at the Hardware Node in the Experiment Window To view or edit the current hardware configuration select the Hardware Node in the Tree View You can expand the node to see what components are currently included by clicki
6. Simple Batch Experiments Troubleshooting My Intensity readings are lt 500 Cnt s The dark count for the photodiode detector ranges from 100 to 500 Cnt s depending upon the DynaPro model If you are continuously getting non zero Intensity readings that are lt 500 PROTEINSOLUTIONS www protein solutions com 56 Cnt s the indication is that the laser light is not reaching the detector You should first check the following 1 Insure that the opaque side of the cell is face the left side of the MicroSampler as you re facing the unit 2 Insure that the lid of the MicroSampler is closed If you are still getting low counts the fiber optic cables may be incorrectly connected 1 Disconnect the power to both the DynaPro host unit and the MicroSampler 2 Review the Connecting the optics block section in the Help file or the Connecting Fiber Optics page in the DynaPro QuickStart Manual 3 Check both fiber optic connections to insure that the notch on the fiber is properly seated in the groove of the connector and that the collar is tightened such that there are no threads showing 4 Reconnect the power to the DynaPro host unit and MicroSampler If you are still getting Intensity readings on the order of the dark count contact the Support Department at Protein Solutions Inc 1 888 242 2848 to report the problem PROTEINSOLUTIONS www protein solutions com 57 Using DYNAMICS V6 PROTEINSOLUTIONS www protein
7. and USB devices M Serial COM 2 empty easurements empty UltroSpec 4000 Connect Titrator Disconnect TC Unit The hardware configuration for the current experiment is defined in the properties table for the Hardware Node The list boxes in the Value column are used to change hosts and or optics blocks assign external detectors to the analog jacks only available if a Flow Kit was included and assign RS232 devices to the serial COM Ports on your PC e g the TC unit for temperature controlled MicroSamplers Once the hardware configuration is defined the Connect button is used to connect the DynaPro host unit and open communications between the software and the various hardware devices Note that you can view and edit selected properties for a given piece of hardware such as the absorbance wavelength for an attached UV detector by selecting the appropriate branch of the Hardware Node in the Tree View Notes amp Tips 1 If you routinely use a specific hardware configuration the File Save Settings command on the main menu bar can be used to save the current configuration as the default such that it is always loaded when a new experiment is started PROTEINSOLUTIONS www protein solutions com 47 2 If you wish to save the current settings for quick retrieval at a later date use the File Save PreSet option on the main menu bar The settings can then be retrieved using the File Open PreSet option
8. Auto Detect COM Ports i Dynamics Y6 has determined that the COM port settings for this experiment are inconsistent with the PC connections The Hardware Node settings have been updated to reflect the correct RS232 connections Setup Troubleshooting PROTEINSOLUTIONS www protein solutions com SAMPLE PREPARATION PROTEINSOLUTIONS www protein solutions com Sample Preparation Overview The objective of the Sample Preparation tutorial is to introduce the new DynaPro user to some of the tricks of the trade with regard to preparing samples for light scattering measurements As you ll soon discover the light scattered from a solution of particles is very sensitive to sample impurities and dust The development of routine procedures for preparing samples and collecting measurements is imperative for success This is not to say that successful light scattering studies are an impossibility They simply require a little more attention in areas considered somewhat irrelevant in other laboratory instrumental techniques With experience you ll discover your own recipe for the perfect sample preparation technique What we will attempt to give you here is a foundation upon which you can built Good luck with your studies and please feel free to contact us should you encounter problems Cleaning the cell Light scattering measurements are extremely sensitive to the presence of dust and or non miscible impurities in the sampl
9. Host Serial Number Used to distinguish and identify host units Optics Serial Number Used to distinguish and identify optics blocks Serial Com Identifies the serial COM port used for communication with DYNAMICS V6 support devices such as a temperature control optics block a titrator and a DLS plate reader Host Optics Node Analog Digital I O True or False indicator for whether or not the host unit includes BNC connections for A D input and output Flow Kit Upgrade True or False indicator for whether or not the optics block includes the 2 front mounted threaded capillary tubing connection ports Host Model Identifies the host model Options are 99D with two USB PC connections and 99E with a single USB PC connection Internal Laser True or False indicator for whether or not an internal laser is contained within the host and or the optics unit Laser Wavelength nm The laser wavelength in nanometers This parameter can be found in both the Host and or Optics Nodes but is only available if the Internal Laser property is set to True Optics Model Identifies the optics model Current options are the MS 800 for small particles at low concentration the MS X for larger particles at low concentration and the LSR for larger particles at higher concentrations Stir Kit Upgrade True or False indicator for whether or not the optics unit includes a stir pad for sample stirring Optics units with Stir Kit Upgrades can be d
10. Rtr lax 589nm amp 20C 1 33 Sample viscosity 20c op 1 019 UserDefined Cauchy Coefficient nm 3119 E Measurements lial Temperature Model Aqueous i ae Solvent Intensity Cnt s 0 Measurement Click here to define sample parameters Ln Expt Hardware Value Parameters Globular Proteins Fixed Instrument Cone mg mL 0 Vol mL 0 dnidC CmLig 0 187 Tol Int Cntis 100000 UserDefined Measurements Measurement Next 7 Sample parameters such as the MW R Model used to estimate the molecular weight the concentration the sample volume the refractive index increment dfi dC and the scattering intensity of a toluene standard are defined in the Sample sub node The use of these parameters in the various calculations within DYANAMICS V6 is detailed in the Application Oriented Software tutorial The User Defined sub node is used to store parameters and values that may not be common to all experiments User defined parameters along with their units are defined in the Tools Parameters User Defined window accessed from the main menu bar To add a parameter to the experiment select the parameter from the drop down list box click the Add button to add it to the property table and then enter the value for the selected measurement in the appropriate value cell PROTEINSOLUTIONS www protein solutions com
11. Selection Boy Intensity Cnt s X 1 00e 003 o R nm Rescale z Click here to show or hide the legend For Help press F1 NUM Ui Notes amp Tips 1 The figure in the Trace View can be modified to user preference using the following keyboard key and mouse button combinations Ctrl both buttons to shrink or enlarge Alt left button to zoom and Shift both buttons to move The Rescale button in the Control Panel will restore the default figure settings Marking outlying data points In the screen shot of the data results shown in the figure below both the Intensity and the Radius data indicate a spike during the third acquisition shown at the 30 second mark on the X axis The other 9 acquisitions indicate constant values for both the Intensity and the Radius suggesting that a dust particle may have momentarily entered the laser beam path during the third acquisition time period If all ten acquisitions are considered for averaging purposes the mean Intensity and Radius values for Measurement 1 would be skewed due to the presence of a single outlier For low MW and low concentration samples the standard approach of re measuring the sample in the hopes of collecting a better data set could be problematic particularly for samples with time dependent properties In the DYNAMICS V6 software the user has the option to Mark outliers thereby removing them from subsequent calculations and graphical display Outlying data is Marked usin
12. The Auto Adjust Laser Power and Real Time Data Filter functions are activated or deactivated in the Fixed sub node and are discussed in the Using DYNAMICS V6 tutorial PROTEINSOLUTIONS www protein solutions com 48 The Instrument Solvent Sample and User Defined sub nodes contain variable parameters i e parameters that are measurement specific All variable parameters associated with instrument control sample acquisition time reading interval and laser power are contained within the Instrument sub node DYNAMICS V6 Exp1 O x fy File View Tools Window Help Oe Expl Hardware Parameters Fixed Solvent Sample UserDefined Measurements Meas 1 Meas 2 Acq Time s Read Interval s 1 Laser Power For Help press F1 DYNAMICS V6 Exp1 O x fi File View Tools Window Help la x Den Sample acquisition time E wj m af for measurement 2 Expl Hardware Parameters Acq Time s Fixed Read Interval s Solvent Sample UserDefined Measurements Meas 1 Meas 2 Laser Power For Help press F1 The solvent refractive index and viscosity are required parameters for calculating the hydrodynamic size from dynamic light scattering data To define the solvent in DY
13. B 2140690 B 49709 0 476 0 009 4 16e 007 2 20e 008 5 1 0 3 Cancel 0 000 For Help press F1 Two special right click menus that can be accessed from within the Grid View are the Column and Units Menus The Column Menu is accessed by right clicking the property i e the top row in the column From this menu the column can be removed from the Grid View display or the Format Data window can be opened Alignment and significant digits for the data within the column are handled from the Format Data window The Units Menu for a given property is accessed by right clicking the Units cell for that column A variety of units are supported in DYNAMICS V6 including standard units for length volume time diffusion intensity mass temperature molecular weight voltage and concentration both weight and number Graphical displays in the Trace View The Trace View gives a graphical representation of the experimental data The display can be changed to the Trace View by clicking on the Trace View button on the Experiment Window menu bar The figure in the Trace View can be displayed in a multitude of user defined formats via the options available in the Control Panel which is displayed by clicking the Control Panel option in the Trace View right click menu The data set shown in the following screen shot was collected with a DynaPro MS X Molecular Sizing Instrument The parameters to be displayed were defined using the X a
14. Notes amp Tips 1 The right click menu Data Filter option is also active while the Experiment Window is in the recording mode It is recommended however that you use it sparingly since the routine can tend to slow the system down if you re attempting to filter a large data set especially if your filtering limits include fluctuations See Real time data filtering for an alternate approach that is better suited for data filtering while in the recording mode Real time data filtering Even though the Data Filter can be accessed and applied while the Experiment Window is in the recording mode it is not recommended that the Data Filter be used while you re PROTEINSOLUTIONS www protein solutions com 87 collecting and recording data For large data sets the routine tends to slow the system down especially if you re applying fluctuation limits An alternate approach is to set the Real time data filter option in the Options Table Tools Options on the DYNAMICS V6 main menu to True This option enables a special form of the Data Filter routine that is better suited for filtering data in real time When the Real time data filter option is enabled only the Maximum and Minimum limits defined by the user in the Data Filter are applied while the Experiment Window is in the recording mode Application of any fluctuation limits is deferred until the data recording is stopped At that point all the limits enabled in the Data Fil
15. and deleted using the Edit Solvents window accessed via the Tools Parameters Solvents option on the main menu bar To define a new solvent select the New option in the Solvent list box and then enter the appropriate parameters include a name for the new solvent After entering the parameter values the Save button can be used to save the new solvent to the Solvent Database with a User Defined Parameter Source Solvents are deleted from the Solvent Database by selecting the solvent in the Solvent list box and then clicking the Remove button DYNAMICS Y6 _ O x File View Tools Help Options Click here to select a solvent for removal or Calculations gt Solvent Rrsuesossunsssnussnnssnssuussunsnunssunssusssussunsel Name SDS mix 01 Bal Parameter Source User Defined Rtr Idx 589nm amp 20C 1 425 Viscosity 20C cp 1 05 u Cauchy Coef nm 3119 Tamn hindal Aqueous Edit properties and then Save to Solvent Database Save Remove Defining user defined parameters While user defined parameters can be added to the experiment at the User Defined Node in the Tree View only those that are currently defined can be added at this level New user defined parameters are defined and saved to a Parameter Database in the Edit User Defined Parameters window accessed using the Tools Parameters User Defined option on the main menu bar To add a user defined parameter to the database enter the p
16. and green in all other views Note that if you have selected the Apply to all measurements option the All data and Selected data options are disabled If you have Marked data manually the Clear manual marking and Keep manual marking options can be used to either discard or keep the previous manual marking this includes manual marking in other measurements if the Apply to all measurements option is also selected Once the limits are defined click the OK button to initiate the automated Data Filter routine dynamics MSX qa3 exp olx PY Eile Edt View Window Help la x ee O Finer Seuings BJ 7 Dynamic Data After enabling the filter option enter the filtering limit here Mark any Intensity readings that deviate from the mean by gt 10 Enable Minimum Amplitude m Maximum CN NO Maximum Amplitude F Amplitude Fluctuation p Baseline Limit 1 p 002 m Options i 140 rear I Apply to all measurements 505 Fluctuation p Alldata Cc C Selecteddata Mark all correlation data ForHt with SOS values gt 40 Cancel Apply the filtering criteria to all data in the current measurement Intensity Fluctuation 10 Data Filter Ctrl F Figure Settings Control Panel Statistics u u U i DynaGraph 4 Use these options to either keep or clear data marked via the right click menu
17. and in the Trace View are alphabetized Auto Save Workspace True or False descriptor indicating whether or not the user would like DYNAMICS V6 to automatically save the workspace settings to the control file when the software is closed Calculate Polydispersity True or False descriptor indicating whether or not the Cumulants polydispersity calculations are to be performed Connect On File New True or False descriptor indicating whether or not DYNAMICS V6 will automatically Connect the instrument when a new file is opened Font Size The font size for the Grid View Instrument Control History Length The number of instantaneous readings displayed during data monitoring with the Instrument Control Panel Y Axis Autoscaling The percentage of 1 the maximum data value added to the maximum and 2 the minimum data value subtracted from the minimum to determine Y axis scaling limits in the Trace Correlation and Regularization Views Using DYNAMICS V6 Troubleshooting PROTEINSOLUTIONS www protein solutions com 96
18. at the Measurement level CNF calculations and baseline adjustments Notes amp Tips 1 Outlying data points can also be Marked in real time Because the figure is continuously updating however real time data marking is easier in the Grid View 2 In DYNAMICS V6 if any parameter associated with a correlation curve is considered to be an outlier then the correlation curve itself is consider to be bad If you Mark a single parameter derived from a correlation curve all other parameters derived from that same correlation curve will also be marked Parameters associated with a correlation curve are radius R polydispersity Pd polydispersity Pd polydispersity index PdI amplitude baseline SOS and MW estimated from the hydrodynamic radius MW R PROTEINSOLUTIONS www protein solutions com Using the Data Filter The DYNAMICS V6 Data Filter is an automated routine for marking data outliers according to user defined limits The Data Filter can be accessed from the right click menu for any of the Measurement Node views Grid Trace Correlation and Regularization To apply a filtering limit enable the limit by checking the Enable box and then enter the limit into the text box Multiple filtering limits can be applied simultaneously Use the All data and Selected data options to apply the filter to either all the acquisitions in the measurement or just the selected data highlighted in blue in the Grid View
19. be resolved The Resolution Slider is included in the Regularization View Control Panel to facilitate a small measure of peak resolution control in cases where apriori information is available The Regularization algorithm is a non linear fitting routine that maximizes the randomness of the residual the difference between the fitted and the measured correlation curve The Optimal position on the Resolution Slider is the resolution that achieves this maximum If it is known however that the peak is comprised of multiple particles moving the Resolution Slider to the right will relax the limits on the randomness of the residual and enhance the possibility of resolving the particles Notes amp Tips 1 The figure in the Trace View can be modified to user preference using the following keyboard key and mouse button combinations Ctrl both buttons to shrink or enlarge Alt left button to zoom and Shift both buttons to move The Rescale button in the Control Panel will restore the default figure settings Formatting tables There are 3 types of data tables in DYNAMICS V6 the Grid View table the Results Summary table in the Regularization View and the Statistics Table Each of these tables can be formatted to user preferences using the Table Settings worksheet The worksheet is opened by selecting the Table Settings option on the table s right click menu Current column and Right click here to unit format for table remove a co
20. be expanded into 5 sub nodes Fixed Instrument Solvent Sample and User Defined Parameter values are entered by selecting the appropriate Parameters sub node in the Tree View double clicking the property value cell and entering the new value or selecting the new value from the list box Aside from the parameters in the Fixed Node all experimental parameters can be varied between measurements using the Measurements list box at the bottom of the property table In addition to viewing in the node specific property table parameters contained within the Instrument Sample and User Defined Nodes can also be displayed in both the Grid and Trace Views Example screen shots of the Fixed Instrument and Sample Node properties are given below see the Selecting a solvent and User defined experimental parameters sections of the tutorial for descriptions of the Solvent and User Defined Nodes respectively Definitions of each property are contained within the DYNAMICS V6 parameters section of the tutorial PROTEINSOLUTIONS www protein solutions com 76 Exp Fee ef ewe a El Expt H Hardware Parameters Auto Adjust Laser Power gred Real Time Data Filter Instrument i Solvent Ignore the 1st Channels Truncate at Channel Cell Constant El Expt E Hardware Acq Time s Read Interval s Laser Power SE Solvent Double click to edit the value E Expl E me E Expt H Hardware E Paramet
21. cms nm E Measurements 7 Acq1 10 2252930 0452 3 69007 4 man 75 OMB T2 acq 2 2222120 040 3 796007 Mak M 73 Acq 2 3 Acqg3 30 2132060 0 480 4 10e007 Copy chee 57 Acq 3 4 Acq4 40 2116740 nunn o sanoo pe Export 53 Click here to Pae 5 As 51 2122 Data Fiter CuhE 57 show readings fee 6 mege 61 2109 Click Menu Table Settings Ct S 50 Acq 7 Acq 71 20984 u v Show Statistics 45 4cq8 8 Acq ta MAMAN TT DEE an ya a g Acqgo 9 q 10 Acq10 101 Item Time Intensity Amplitude Baseline SOS _ BS Cnt s Ag O 56 Read1 21 2252930 ao Read2 22 2222120 es Read 3 23 2132060 BB 3 4 Read4 24 2116710 eme spies il IS ReadS 25 2122260 6 Read6 26 2109050 7 Read7 27 2098470 8 Read8 28 2120440 9 Read9 29 2124220 140 Read 10 30 2108640 The average and standard deviation for each column excluding Marked data are given in the Statistics Table The right click menu for the Grid View is accessed by right clicking on one of the white background cells in the table Brief descriptions of the available commands on the right click menu are given below Mark This command is used to mark a piece of data as an outlier that is to be excluded from all calculations Marked data in the Grid View appears in red Copy This command copies selected data to the Windows clipboard Copied data can then be pasted into other Windows applications such as Excel Word and or Power Point
22. detail see Theory Of Operation The DynaPro determines the uniformity of sizes through a monomodal single particle curve fit analysis called Cumulants which assumes a single particle size with a gaussian distribution The SOS is the Sum Of Squares difference between the measured and the Cumulants calculated intensity correlation curves and is reported for each sample acquisition a single correlation curve in the Grid View of DYANAMICS V6 Low SOS values lt 20 indicate reasonable agreement between the measured correlation curve and the Cumulants fitted curve and suggest the sample is likely monomodal with low polydispersity i e a tight size distribution Some samples will not be monomodal but will contain multiple populations of different sizes For these samples you should expect to observe increased SOS values e g gt 100 remember that the Cumulants algorithm forces a single particle fit If the sample appears to be multi modal the size distribution histogram can be generated using the Regularization algorithm which makes no assumptions regarding the number of size populations Temperature Controlled MicroSamplers The temperature controlled TC instrument actively controls the temperature within the MicroSampler The range of temperature control is 4 to 60 C The temperature can be maintained indefinitely between these extremes with an accuracy of better than 1 C The temperature controlled system uses a Pelti
23. for a complete measurement or set of acquisitions Ideally the solvent intensity should be used for the CNF calculations In the absence of a solvent measurement however the clean water count measured for your DynaPro and reported in the Dynamics ini file can be used as a solvent approximation in the CNF calculations To use the clean water count as an estimate of the solvent check the appropriate box in the Correlation Noise Factor frame on the Instrument Control Panel When adjusting the laser power and acquisition time settings bear in mind that you want both the CNF and Optimal Acq to be as low as possible Recording data Data recording in DYNAMICS V6 is controlled using the Record button on the Experiment Window tool bar For batch mode experiments the Record button exists in 3 states representing the idle mode gray button monitor mode green button and recording mode flashing red button for the Experiment Window For automated experiments a yellow flashing button representing the wait mode is also used Record Button in Idle Mode Monitor Mode Recording Mode Connect Click an s Disconnect fen efa E Expl E Hardware Host 99 50 Optics 045 UltroSpec Intensity Cnt s Temperature Absorbance C m Connecting the instrument will place the Experiment Window in the monitor mode and change the Record button color to green indicating that the system
24. ready to begin recording data To start recording data click the green button The button face will change to a flashing red indicating that DYNAMICS V6 is recording data and incoming data will be displayed in the Grid View of the Experiment Window As data is recorded into the Grid View the mean and standard deviation for each column are reported in the Statistics Table When you feel you ve collected a sufficient amount of data the recording can be stopped by clicking the PROTEINSOLUTIONS www protein solutions com 51 flashing red Record button The button face will then change back to green indicating that software is ready to begin recording data for the next measurement Esper Expt Hardware Time Temp Intensity Baseline Parameters s kCnt s Measurements Notes amp Tips 1 While DYNAMICS V6 is in the recording mode incoming data can be displayed in any of the views available on the Experiment Window tool bar Grid View Trace View Regularization View or Correlation View To change the view click the appropriate view button on the tool bar Setting up the Grid View The Grid View in DYNAMICS V6 is fully customizable using the various right click menus for the table To remove a column or format the data in the column precision and or alignment right click the column header top row in table To access the Units menu for a particular column right click the
25. the solvent at the laser wavelength of the instrument Name The name of the solvent selected for the designated measurement Parameter Source The source of parameter data for the designated solvent The 2 value options are Standard and User Defined where Standard indicates that the solvent parameters were determined from CRC data contained in the Solvent Database Rfr Idx 589nm amp 20C The refractive index of the solvent at 20 degrees Celsius using a 589 nm light source Solv Int Cnt s The measured solvent scattering intensity in units of counts second The value is subtracted from the sample scattering signal to determine the residual intensity of the analyte in static light scattering applications Temp Model The temperature model used to estimate the solvent refractive index and viscosity at temperatures other than 20 C Viscosity 20C cp The viscosity of the solvent at 20 C in units of centipoise Sample Parameters Node Analyte Inj Mass mg The milligram mass of analyte injected onto the column in HPLC applications The value is used to calculate protein extinction coefficients from wavelength dependent UV data and for real time analog RI detector calibration This parameter is only available if an HPLC pump is present in the hardware configuration Conc mg mL The concentration of the sample in units of mg mL The value is used for static light scattering applications such as absolute molecular wei
26. the DynaPro is connected If the connection process fails the Connection Status box will report that the software could not connect Notes amp Tips 1 If you encounter problems connecting check the power if you have a temperature controlled unit both components have power switches and the USB connections If you have a temperature controlled unit insure that the RS232 connections are complete Should you still encounter connection problems contact the technical support staff at Protein Solutions 1 888 242 2848 Auto detecting COM port settings DYNAMICS V6 communicates with the DynaPro host unit using RS232 communication protocol The software also supports a variety of external devices that require RS232 COM port connections In order to communication with RS232 devices the COM port identifier e g COM 1 COM 2 etc for each device must be know During the connection process DYNAMICS V6 will query the available COM ports on your computer and automatically determine the COM port identifier for each of the RS232 devices If configurations different PROTEINSOLUTIONS www protein solutions com 30 from those defined in the Hardware Node are detected the user will be notified of the changes being made Note that COM ports cannot be queried successfully if the RS232 device is not powered Once the correct COM port settings are determined DYNAMICS V6 will automatically save the configuration to the Dynamics ini control file
27. to the PC via the USB cables Windows will detect new hardware open the Add New Hardware Wizard and search for the required drivers Most PC s will find the DynaPro drivers and install them automatically If not you may need to search for the drivers manually Click the Browse button in the Add New Hardware Wizard and find the folder App path Protein Solutions Drivers where App path is the path you selected during the software installation App path c Program Files for typical installations Then use the Next and Finish buttons to complete the process Depending upon the DynaPro model you may need to repeat the process for other drivers Alternately you can point the Add New Hardware Wizard to the DynaPro Drivers and QC Files floppy disk that was shipped with your instrument Add New Hardware Wizard Windows will search for new drivers in its driver database on your hard drive and in any of the following selected locations Click Next to start the search IV Floppy disk drives T CD ROM drives 7 Microsoft Windows Update IV Specify a location App Path Protein Solutionsi Drivers pa Browse Notes amp Tips 1 If you are using a USB hub drivers for the hub can be found on your Windows Installation CD Direct the Add New Hardware Wizard to the Windows CD and the driver will be automatically installed 2 Ifyou are upgrading from Win 98 or Win ME to Win 2000 Windows may not be able to find
28. tool bar The Record button face will then change to a flashing red indicating that the software is in the recording mode To stop recording data click the flashing red Record button Cumulants and Regularization analyses are applied automatically the results of which can be viewed by selecting the appropriate view button on the Experiment Window PROTEINSOLUTIONS www protein solutions com DynaPro Operations The sample is illuminated by a semi conductor laser of 830 nm wavelength The laser light passing through the cell is guided into a beam dump An interlock switch automatically disables the laser if the MicroSampler lid is opened when the laser is operating The light scattered by the sample in the cell is collected and guided via a fiber optic cable to an actively quenched solid state Single Photon Counting Module SPCM The photons are then converted to electrical pulses and correlated The DynaPro analyses the time scale of the scattered light intensity fluctuations by a mathematical process called autocorrelation To perform the very fast data manipulation necessary to obtain results in real time the DynaPro uses the latest generation of correlator running special algorithms The translational diffusion coefficient D of the molecules in the sample cell is determined from the decay of the intensity autocorrelation data The hydrodynamic radius R of the sample is then derived from D using the Stokes Einstein equation for more
29. units cell 274 row in table for that column Right click here to remove a column or format data Amplitude Raseline SOS Remove Column Format Data Mark Copy Right click here to open Table Settings worksheet N ee nos CHS Hide or show Statistics Table by clicking here v Show Statistics PROTEINSOLUTIONS www protein solutions com Columns are added to the Grid View in the Table Settings window To access the Table Settings window move the mouse cursor to the table proper any non header non units location in the table right click and then select the Table Settings option from the popup menu Note that you can also show or hide the Statistics Table from this popup menu Click and drag to move a column x Font Size fia z tem Time Intensity Amplitude D R Pd Baseline sos Right click here to ent Remove Column open Units menu Format Data Options Statistics Rows Edit Options View Options Temperature Current column and unit format for table in selected view Right click here to remove a column or format the data Table Settings Select view type here Mean 5 5 Add Grid View Regularization View Add All a Statistics Table Remove All cen Current rows for Statistics Table Right click to remove T
30. AMICS V6 some terms to remember are Readings instantaneous data collected every second e g the absorbance refractive index and or scattering intensity of the sample at a given moment in time Acquisitions Acq a set of readings and the intensity correlation curve collected across a user defined acquisition time Measurement a set of acquisitions Experiment a set of measurements Launching DYNAMICS V6 The DYNAMICS V6 software package is opened by double clicking the icon on the Windows desktop or single clicking the Start Programs Protein Solutions Dynamics V6 icon on the task bar Start button DYNAMICS Exp1 File Edit View Window Help Cel BR RACK A For Help press F1 The main tool bar in DYNAMICS V6 holds a collection of short cut buttons for performing various tasks The buttons displayed and their locations on the tool bar can be customized to user preferences using the Edit Toolbar option on the main menu bar PROTEINSOLUTIONS www protein solutions com 61 Open new Experiment File Copy selected About DYNAMICS V6 text or figure Experiment File Doel gt S A48 current settings Open saved Experiment File Structure of DYNAMICS V6 The main window in DYNAMICS V6 is a Multi Document Interface MDI window An MDI window functions as a mini operating environment allowing the opening and closing of multiple child windows from within the MD
31. After seating the cell in the DynaPro optics block close the lid to de activate the laser power safety kill switch PROTEINSOLUTIONS www protein solutions com Notes amp Tips 1 Whatman Anotop disposable filters are unidirectional Pulling a sample the wrong way through the filter can break the membrane loose from the housing making it ineffective for sample filtering Slight backward pressures are OK i e for controlling the drip rate but loading the syringe with the filter in place is not recommended 2 If you encounter bubble problems try slowly pulling the needle tip upwards as the sample is dispensed 3 The bowl at the top of the cell window tends to collect dust It is recommended that you place only enough sample in the cell to fill the window Loading the Microsphere PreSet When DYNAMICS V6 is first installed the default experimental settings are those suggested for measuring the Microsphere standard To begin an experiment open DYNAMICS V6 and select the File New option on the main menu bar Alternately if you suspect that the default settings have been changed you can open a new Experiment Window with the Microsphere experimental parameters pre loaded by opening the QuickStart PreSet To open the QuickStart PreSet click the File Open PreSet option on the main menu bar select the QuickStart pst file located in the PreSets folder and then click the Open button Open 2 x Lookin Preses d AMT
32. Batch experiment parameters The parameters describing the experiment are defined in the Parameters Node of the Tree View To expand the Parameters Node into various subcategories click the sign to the left of the Parameters label The descriptors for the experiment can then be entered in the property table for each sub node To enter or edit a value double click the value cell enter the value and then move to another cell or use the Enter button on your keyboard DYNAMICS V6 Exp1 O x Click here to expand peal 1B the Parameters Node Define the experimental parameters here DYNAMICS Y6 Exp1 fi File View Tools Window Hel Expl Hardware For Help Parameters Fixed Real Time Data Fitter Instrument Ignore the 1st Channels 2 Solvent Sample Truncate at Channel 120 UserDefined Cell Constant 1 85 Measurements For Help press F1 The Fixed sub node contains parameters that are applied to all measurements within the experiment The Ignore the 1 Channels and Truncate at Channel parameters are used to define the range of correlator channels used when calculating the analyte hydrodynamic properties The Cell Constant is an instrument calibration parameter used for concentration dependent static molecular weight studies see Application Oriented Software
33. I window The child windows within the MDI window can be moved arranged minimized and or maximized to whatever degree is suitable to the user DYNAMICS MDI Window Instrument Control Panel DYNAMICS Exp1 Jol x File Edit View Window Help Experiment Window OSH teel R 4 Instrument Contol Panel Ess p Data Monitor Start Stop msa N Acquision Time E Expl Hardware Parameters Measurements 4g Wizard Checklist x Sample Name DP Wavelength nm Acquisition Time s O New experiment Collect measurements M Solvent Viscosity cPoise V Solvent Refractive Index History Length fiz For Help press F1 There are 3 child window types in DYNAMICS V6 the Instrument Control Panel the Experiment Window and the Worksheet The Instrument Control Panel is used to verify PROTEINSOLUTIONS www protein solutions com communications with the DynaPro and other external devices set basic parameters e g laser power and baseline value for external detectors and monitor data input The Experiment Window is used to setup run and record save data for new experiments and to view parameters and results of past experiments Data recording saving to memory occurs from within the Experiment Window Worksheets are used to facilitate special calculations and unique tasks within the DYNAMICS V6 MDI environment Op
34. Line Filter A ia me Notes amp Tips 1 Light scattering detectors are much more sensitive to the condition of the run buffer than typical HPLC detectors such as differential refractometers and UV detectors It is strongly recommended that you utilize a degasser and an in line filter 0 5 um frits work well if you are setting your DynaPro system up for chromatography experiments The best location for the in line filter is between the pump and the injection valve Identifying the connection and communication ports The DynaPro and DYNAMICS V6 support a variety of communication protocols and hardware configurations The relevant connection ports on the DynaPro host unit and optics block are shown in the figure below Details on each type of connection are given in the text following the figure Connection instructions and enlarged figures for each type of port cable are given in the appropriate sections of the tutorial PROTEINSOLUTIONS www protein solutions com DynaPro host unit Laser fiber optic port Temperature controlled MicroSampler TC unit BNC Connections The BNC jacks are used to send and receive signals from external devices that support analog digital input and or output such as HPCL detectors injectors and pH meters Note that the DynaPro is specified for positive potential inputs only Negative potential inputs can damage the I O board Please contact Protein Solutions if you
35. M Peak2 62 23 79 46 Results Summary For Help press F1 NUM The Control Panel in the Regularization View is used to define the histogram display parameters define the model to be used for the Intensity to Mass conversion and define the resolution applied in the Regularization algorithm To access the Control Panel right click the figure and check the Control Panel option in the popup menu The Results Summary table in the Regularization View gives the numerical properties of each peak in the histogram and allows the user to remove any nonsense or noise peaks from both the figure and the Intensity Mass calculations To access the Results Summary right click the figure and check the Results Summary option in the popup menu To remove a peak uncheck the option box for that peak in the Item column To change the units for a column remove a column format the data in a column or access the Table Settings worksheet right click the appropriate cell in the Results Summary table see Using DYANAMICS V6 Formatting tables Notes amp Tips 1 The Resolution Slider can be used to change the resolution applied in the Regularization algorithm However this function should only be used if apriori information is available In other words if you know beyond a shadow of a doubt that there are two particles displayed as a single peak in the histogram you may be able to resolve the particles by moving the Resolution Slider to the right If you
36. NAMICS V6 select the Solvent Node in the Tree View on the left side of the Experiment Window and then select a predefined solvent from the Solvent Name list box in the properties table When a solvent is selected the parameters for that solvent will be loaded into the table Note that the refractive index viscosity Cauchy coefficient and temperature model are pre determined and locked for all standard solvents defined within DYNAMICS V6 If you wish to vary these parameters you can select the User Defined option in the drop down list box and then enter your own values for the solvent properties New solvents both standard and PROTEINSOLUTIONS www protein solutions com 49 user defined are defined and saved to the Solvent Database in the Tools Parameters Solvents window see the Using DYANAMICS V6 tutorial Protein Solutions includes a Microsphere size standard with DynaPro deliveries for in field testing and instrument setup If you are using the Microsphere standard you should select Phosphate Buffered Saline PBS as the solvent If you are using an aqueous protein standard such as BSA or lysozyme PBS is also a reasonable solvent choice Click here to open the Solvent Properties Table meea Exp Click here to select the solvent Hardware Property Value El Parameters Name PBS Fixed Parameter Source Standard Baul L
37. Pro optics block Optics Model Defines the model of the DynaPro optics block Currently available optics block models are the MS 800 for smaller particles at low concentrations the MS X for larger particles at low concentrations and the LSR for larger particles at high concentrations Laser Wavelength nm Defines the laser wavelength in nanometers for either the host unit IFlex True or the optics block IFlex False Note that in earlier versions of the Dynamics software the laser wavelength was entered in units of Angstroms A TC Upgrade Identifies whether or not the DynaPro optics block includes temperature control facilitated through an RS232 PC connection Stir Kit Upgrade Identifies whether or not the DynaPro optics block includes a magnetic stir pad for sample stirring Optics blocks with Stir Kit upgrades include a single need insertion port in the center of the lid on the top of the optics block Flow Kit Upgrade Identifies whether or not the host and optics block include a Flow Kit which facilitates flow mode experiments such as automated titrations and HPLC Units with Flow Kit upgrades can be identified by the presence of 6 analog digital BNC connections on the back of the host unit and 2 threaded capillary tubing connection ports on the face of the optics block UltroSpec 4000 Identifies whether or not an UltroSpec 4000 UV spectrophotometer is available for integration Titrator Identifies
38. TEINSOLUTIONS www protein solutions com 12 Fixed Parameters Node Auto Adjust Laser Power True or False indicator for whether or not the DynaPro laser power is automatically adjusted to optimal settings during the course of an experiment Note that when the laser power is optimized automatically the current measurement is stopped and a new measurement is started Cell Constant The instrument calibration constant for static light scattering applications The parameter is integrated into the Rayleigh equation as a Rayleigh ratio multiplier to compensate for non ideality in the polarization maintaining fiber optics Ignore the 1 Channels The number of initial channels in the DynaPro correlator that are to be ignored in the Cumulants and Regularization algorithm analysis of the dynamic light scattering correlation curve Real Time Data Filter True or False indicator for whether or not the data filter algorithms are applied in real time i e while the data is being collected Titrant Conc mg mL The concentration in mg mL of the analyte in the titrant In dilution type titrations the analyte can reside in either or both the sample and the titrant solutions Truncate at Channel The final correlation channel to be considered in the Cumulants and Regularization algorithm analysis of the dynamic light scattering correlation curve Instrument Parameters Node Acq Time s The amount of time in seconds taken to collec
39. To connect the instrument select the Hardware Node in the Tree View and click to Connect button below the properties table Once the instrument is connected the Record button on the Experiment Window tool bar will turn green indicating that the software is ready to begin recording data To start recording data for the Microsphere standard click the green Record button on the tool bar The button face will change to a flashing red indicating that DYNAMICS V6 is recording data and incoming data will be displayed in the Grid View of the Experiment Window As data is recorded into the Grid View the mean and standard deviation for each column are reported in the Statistics Table After collecting 10 acquisitions Acq under Item in the data table stop the data recording by clicking the flashing red Record Button The button face will then change back to green indicating that software is ready to begin recording data for the next measurement PROTEINSOLUTIONS www protein solutions com 41 DYNAMICS amp File View Jools Window Help 18 x EFI Em jaj coli Exp1 Hardware Time Temp Intensity Parameters kCntis Measurements Statistics Table For Help press F1 NUM 7 The APD detector in the DynaPro can be permanently damaged if the scattering intensity is too large The Detector Protector function in DYNAMICS V6 is designed to prevent damage to the APD by red
40. User Manual For The DynaPro Molecular Sizing Instrument And DYNAMICS V6 Software Molecular Sizing Instruments Contact Information Protein Solutions 750 Vassar Ave Lakewood NJ 08701 USA UK 1 732 560 8550 Phone 44 1494 446 652 Phone 1 732 370 0032 Fax 44 1494 446 672 Fax PROTEINSOLUTIONS www protein solutions com Table of Contents Technical Manual Statement Of Warranty Statement Of Liability Validity Of This Document Technical Manual Overview DynaPro Block Diagram Setting Up The Instrument Using The DynaPro DynaPro Operations Temperature Controlled MicroSamplers Fluid Cooling Heating Enhancements Remote Dry Air Purging Periodic Maintenance General Notes Precautions And Troubleshooting Setup Setup Overview Installing DYNAMICS V6 DYNAMICS V6 Installation Options Unpacking the DynaPro Stacking the DynaPro modules Titration amp HPLC setup considerations Identifying the connection and communication ports Connecting the optics block Connecting RS232 cables Connecting analog digital I O devices Connecting the USB and power cables Establishing Windows DynaPro USB communication links Starting DYNAMICS V6 Original Hardware Configuration Connecting to the DynaPro Auto detecting COM port settings Setup Troubleshooting Sample Preparation Sample Preparation Overview Cleaning the cell Filtering samples Using the MicroFilter kit Spinning the sample Loadi
41. able in the Control Panel If the Control Panel is not visible right click the figure and check the Control Panel option in the popup menu hr Display Trace View AE Ele Edt view Window Help 18 x DS t helg Select the parameters mfe Lia al 7 to be displayed here E MSX qa3 exp E Hardware is Y Axis Click here to change from line to symbol Mintensity Amplitude D dynamics 22007 Intensity Cntis 2000 E Parameters Temperature Oo Apply a scalar to the MAR Ei selected data set here E Measurements E 24004 1800 V Line 74 0 Scalar 0 001 a a a a a Dune am a zen vum ms zu 2 0 10 20 30 40 50 60 70 80 90 100110 Left Select the display Time 8 Right axis here M Legend Tees Boy Intensity Crit s X 1 00e 003 o R m 17 Cursor Click here to show For Help press F1 M mnom or hide the legend The parameters displayed in the Trace View figure are user definable via the X and Y Axis list boxes in the Control Panel The default display for each parameter is a line To change from a line to a symbol select the parameter either in the Y Axis list box or in the figure legend and then uncheck the Line option in the Control Panel The display axis for a parameter can be changed using the Left and Right radio buttons on the Control Panel The Scalar text box in the Control Panel is used to apply a multiplier to the se
42. allows the instrument to be powered up for extended periods of time before the problem is remedied PROTEINSOLUTIONS www protein solutions com 13 e Ifthe user finds a problem which is not described in this manual or if the suggestions given here don t appear to work please contact a Protein Solutions Technical Representative at support protein solutions com or at one of the following locations Protein Solutions 750 Vassar Avenue Lakewood NJ 08701 U S A Tel 1 732 367 1663 1 888 242 2848 Fax 1 732 370 0032 Protein Solutions Ltd The Upper Floor Liverpool Victoria House High Wycombe HP13 6SF England Tel 44 1 494 446 652 Fax 44 1 494 446 672 PROTEINSOLUTIONS www protein solutions com SETUP PROTEINSOLUTIONS www protein solutions com 14 Setup Overview The Setup tutorial is the first in a series of tutorials developed as training tools for new DynaPro users The tutorial includes step by step instructions for unpacking the instrument connecting the modules and establishing communication links between the DynaPro host unit and DYNAMICS V6 Should you encounter any problems please visit the Technical Support section of our website at www protein solutions com Installing DYNAMICS V6 The installation disk for DYNAMICS V6 includes the drivers in a compressed format for the various USB components within the DynaPro The suggested setup procedure is to install the software pr
43. ame is used to add auxiliary and or USB devices to the hardware configuration Exp1 O x ELA ol a ca Expt Hardware Host test Host Serial Number test r Optics test Optics Serial Number test UV Detector Analog hue empt Test Pump 1 a09 p u ia Parameters __ Analog Input 2 pe Detector Click here to Measurements em r ates pty I select device Serial COM 1 empty Auxiliary HYY 1 Test Pump X Connection Status ic Connected a Connect Disconnect PROTEINSOLUTIONS www protein solutions com Notes amp Tips 1 Communication protocols for USB and RS232 devices are device specific and are hard coded into the DYNAMICS V6 software If you have a particular device or detector that you would like to have supported please contact Protein Solutions and let us know 2 Once an experiment has been conducted the removal and or addition of hardware units is prohibited 3 Hardware can only be selected at the Hardware Node To edit or add new hardware components use the Tools Parameters Hardware option on the main menu bar Setting hardware properties Properties and settings specific to a particular hardware component can be viewed by selecting the appropriate component in the Tree View With the exceptions of the Host and Optics components which are read only the property sett
44. arameter name in the empty property cell at the bottom of the table The list box in the units cell contains a complete list of DYNAMICS V6 supported units Alternately you can define your own units although unit transforms in the Grid View would not be allowed After the units have been selected click the Save button to update the Parameter Database with the new user defined parameter User defined parameters are deleted from the Parameter Database by selecting the entire row containing the parameter to be removed and then clicking the Remove button PROTEINSOLUTIONS www protein solutions com 94 95 DYNAMICS Y6 Parameters gt User Defined Edit User Defined Parameters SDS Conc Lot Reactor temp Incubation time lonic strength Enter new user defined parameter here SOVE Application Options Control and display parameters that are applied throughout the application are viewed and or edited in the Application Options window accessed via the Tools Options command on the main menu bar A brief description of each property follows the figure DYNAMICS Y6 Options Application Options Alphabetize Lists Auto Save Workspace Connect On File New Y Axis Autoscaling Instrument Control History Length Font Size Calculate Polydispersity PROTEINSOLUTIONS www protein solutions com Alphabetize Lists True or False descriptor indicating whether or not the list boxes in the Table Settings window
45. are unsure you should always the default Optimal Resolution PROTEINSOLUTIONS www protein solutions com 55 2 Figures can be copied to the Windows clipboard using the Copy command in the right click menu To copy a figure into a Word document right click the figure select Copy from the popup menu select a location in your manuscript and then select Edit Paste from the Word menu bar or use the Ctrl V shortcut The figure will be pasted into the Word document as a bitmap image Printing a one page summary To print a one page summary of results select the File Print Summary option on the DYNAMICS V6 menu bar The figures and data included on the printed page will be dependent upon the type of experiment conducted The figure below shows an example of a one page print summary for a simple batch experiment where the Hardware Node in the Tree View contains only the DynaPro host unit and MicroSampler DYNAMICS VE Resuks Summary File properties and annotations Tile cane of ne age sunny prt Name MS 800 qa3 exp Exp Date 04 Jan 2001 05 50 04 Comments 122m Microsthvre quality cortroltest for MS 800 Experiment and User Defined parameters 02 19 01 Figure from Trace View ka Har ome re a Laser Power 7a En a Cumulants results from Grid View Size distribution j Ad 0 723 DIO 1 200 DECO Results Summary from Regularization View
46. arization analysis of the correlation curve associated with the selection in the Measurement Node Display PROTEINSOLUTIONS www protein solutions com 64 65 options include Radius Diameter Diffusion Coefficient and Delay Time for the X axis and Intensity Mass and Number for the Y axis Record Button Used to start and stop recording data into an Experiment Window and to start and stop automated experiments History Monitor Opens a worksheet window containing a history of actions events for the current experiment Wizard Checklist Opens a worksheet window containing a list of actions that must occur and or parameters that must be entered prior to enabling of the Record button Instrument Control Panel Displays the Instrument Control Panel for monitoring data and or adjusting control parameters prior to recording data Titrator Control Panel Displays the Titrator Control Panel for instrument initialization syringe cycling and manual injection control Defining hardware New hardware components are defined or existing hardware components edited in the Tools Parameters Hardware window available on the main menu bar DYNAMICS V6 lolx File View Tools Help Solvents User Defined Property Original Equipment Host Serial Number C Host Units Host Model Optics Blocks IFlex Laser Optics Serial Number Optics Model Laser Wavelength nm TC Upgrade Stir Kit Upgrade F
47. cement volume between the DynaPro optics block and the A D detector This property is only available if an HPLC pump is available in the hardware configuration It is used along with the pump Flow Rate mL min to calculate the time difference in the HPLC peak shift routines Wavelength nm The wavelength in nanometers for an analog UV detector Titrator Node Cell Type Defines the type of cell used in a titration experiment Available options are the Flow Cell for titrations of a sample contained outside of the optics block and the Stir Cell for titrations of samples contained within the measuring cell in the optics block Max Sample Vol mL The maximum volume in milliliters of the container holder the sample and into which the titrant is being injected The titrator control module will track the total injection volume to insure the sample vessel is not overfilled Sample Syringe Vol mL The milliliter volume of the sample syringe on the right side of the automatic titrator Syringe Speed s stroke The required amount of time in seconds to either completely fill an empty titrator syringe or completely empty a full titrator syringe Titrant Syringe Vol mL The milliliter volume of the titrant syringe on the left side of the automatic titrator Titration Type The type of titration that is was conducted In a Dilution type titration the analyte can reside in either or both the sample and the titrant solutions PRO
48. d by researchers using light scattering instrumentation The standard argument is that if I filter my sample I m not really measuring the original sample To a very limited extent this argument is correct and if you want to see whether or not you ve got some very large stuff in your sample then you should avoid filtering It must be recognized however that very large stuff includes dust which really isn t an integral component of the analyte If you find yourself questioning whether or not to use a filter remember the following 1 The upper size limit for DLS is dependent upon a variety of factors principle of which is the diameter of the laser beam in the scattering volume If the particles that you re attempting to measure are big compared to the beam diameter you re likely to encounter number fluctuations which means that the number of particles within the scattering volume during the course of the measurement is not constant While the presence of number fluctuations see PSI Books What are number fluctuations for tips on recognizing them will tell you that you have large particles in the sample you will not be able to get a size or mass distribution 2 Dust or non sample contaminants are typically very very large and can usually be removed with a 0 45 um filter For smaller size analytes such as proteins the pore size for this filter is sufficiently large to pass all of your true sample 3 If yo
49. d intensity in your experiment should be within about 10 of the value measured in the QC test The size distribution for the Microsphere standard can be displayed by selecting the Regularization View button on the tool bar The Results Summary table is shown below the histogram The known hydrodynamic radius for the Microsphere is 6 nm with reproducibility of about 0 3 nm PROTEINSOLUTIONS www protein solutions com lull Display Regularization View 99 179 qa3 exp 99 179 ga3 exp l Hardware u Size Distribution Parameters E Measurements 2 40 DE 5 E 20 0 0 01 10 00 1 00E 04 1 00E 07 Radius nm Results Summary Notes amp Tips 1 The Microsphere formulation is sensitive to temperature sample can aggregate While the 0 02 um filter is sufficient to remove large aggregates it will not remove low order X mers e g dimers and trimers shipping conditions at the time your instrument was delivered your Regularization results may indicate a slight elevation in the measure radius 6 3 to 6 8 nm due to the presence of low order aggregates This is no cause for alarm It is a characteristic of the sample rather than the instrument Microsphere Troubleshooting PROTEINSOLUTIONS www protein solutions com ne Control Panel Optimal Resolution X Anis Radius ba Y Axis ln
50. del 99D ial Click here to select a unit A D Detectors g een Laser for editing or to define c A p Laser Wavelength nm 825 a new unit Auxiliary Devices Analog Digital WO True X Optics Block properties Property Yalue Original Equipment Optics Unit 07 Host Unit Serial Number 07 aj Model MS 800 Z m Internal Laser False X C A D Detectors pel 7 ae i TC Upgrade Fale E Change the Serial Number Auxiliary Devices Sti KE Upgrade True i to define a new unit with Flow Kit Upgrade True the same properties Remove Click here to delete a unit from the DV6 control file Click here to save changes The properties for the individual host and optics units differ slightly from those of the original equipment These differences are integrated to allow the mixing of units with differing degrees of Flow Kit and Stir Kit upgrades External analog and digital detectors that are to be integrated with the DynaPro Molecular Sizing Instrument are defined edited and or deleted using the A D Detectors and Auxiliary Devices radio buttons Currently DYNAMICS V6 supports any pH RI differential refractive index or UV detector that generates an output signal between 0 and 2000 mV Supported PROTEINSOLUTIONS www protein solutions com 67 auxiliary and USB devices include an HPLC Pump for DynaPro integration in flow mode chromatographic applications Er O A D Detector properties
51. e cell facing the back of the optics block The capillaries are then attached in the fashion shown in the figure below PROTEINSOLUTIONS www protein solutions com 18 19 Ideally the sample should migrate upward from the bottom of the cell during flow This type of flow is achieved by using the left capillary port as the inlet port and the right capillary port as the outlet port If an automatic Titrator was included with your DynaPro purchase it comes equipped with all the capillaries syringes needles and fittings needed for typical titration experiments along with a supply of dust free septum vials and a vial holder The capillary tubing lengths are kept short in order to minimize the required volume of sample and titrant necessary for automated titrations As such the Titrator needs to be as close to the MicroSampler as possible Capillary connections are more easily handled if the Titrator is placed to the left of the DynaPro as you re facing the system A typical setup scheme for DynaPro coupled automatic titrations is given in the following figure _ Sample Syringe PROTEINSOLUTIONS www protein solutions com 20 In order to minimize band spreading in HPLC experiments capillary tubing lengths should be kept to a minimum A typical setup scheme for DynaPro coupled HPLC experiments is given in the following figure Coupled RI Detector mm 3 a f Besser In Line iter gt es In
52. e solution It is therefore imperative that the light scattering cell be clean and dust free prior to sample loading Recommended cleaning procedures for the quartz cells used in DynaPro instruments are as follows 1 Using a plastic transfer pipette flush the cell multiple times with a mild surfactant or soap solution e g 1 Triton X 100 to remove any dust and or residual impurities from the interior cell surfaces 2 Rinse the cell several times with filtered DI water 3 With the cell in an inverted position dry the interior using compressed air 4 Check the exterior surface of the cell windows and remove any smudges or fingerprints using a clean piece of lens paper Note that tissues and other laboratory wipes can scratch the surface of the cell and are not recommended 5 Using compressed air remove any dust from the surface of the cell cap replace the cap and then set the cell aside until ready for use Notes amp Tips 1 Concentrated acids and bases can etch the cell surface and are not recommended 2 Organic solvents have been known to leave a thin residue on the cell surface Hence the use of acetone or ethanol as a means of drying the cell interior is not recommended 3 Ifthe cell is still not clean after flushing with soap 15 to 20 minutes in a sonication bath will help PROTEINSOLUTIONS www protein solutions com 32 Filtering samples The question of whether or not to filter a sample is commonly raise
53. ening a new experiment file In DYNAMICS V6 new experiments are initiated using either the page icon on the main tool bar or the File New option on the main menu bar When either of these options are used a new Experiment Window is opened within the main window The Experiment Window is used to setup run and record save data for new experiments and to view parameters and results of past experiments Open New Experiment DYNAMICS Exp1 Edit View Window Help 7 su z28 Text boxes for user defined Expl Title and Comments E E E w Hardware DynaPro El Parameters User Defined Calculations Event Schedule Measurements T gt Properties eo 5 File Name Expl Sizing Bar Click and Data Last Modified 27 Jan 2001 17 50 47 drag to resize views Exp File Version 6 05 07k1 27 Jan 2001 17 50 48 Exp Time Stamp Title PS Microsphere standard hydrodynamic radius For Help press F1 The adjustable sizing bar in the Experiment Window separates the window into two sides the list side and the display side The left side of the Experiment Window holds the Tree View which contains a list of categories within which the experimental information and data are grouped The right side of the Experiment Window is used to display the specific information parameters and or data associated with the particular node selected in the Tree View Command functions
54. er cases Periodically wipe down the outside case of the instrument with a clean moist cloth to keep it free from dust or surface stains PROTEINSOLUTIONS www protein solutions com 11 Drying Tube A weekly check on the condition of the desiccant container is recommended As the desiccant becomes progressively saturated with moisture a band of pink will be seen to move from front to rear Earlier DynaPro models will have been fitted with a lid to gain access to view the desiccant Later models 1998 now have 3 viewing windows When 2 of the 3 windows show pink it is time to change regenerate the desiccant The drying tube is located to the side of the instrument beneath the access panel To remove the desiccant container open remove the lid and grasp firmly the container with both hands Lift slowly upwards and separate the container from the two rubber tube holders Unscrew the metal end cap to remove the used desiccant granules They may be replaced with dry granules or re used after drying in an oven Alternatively you can discard the entire desiccant package and fit a new one Replace the new regenerated drying tube in a reverse fashion taking care to align the container correctly as it will only fit one way firmly seated in the connectors It is estimated that desiccant replacement will not be necessary more frequently than every 12 18 months General Notes Precautions And Troubleshooting e The pattern of ventilati
55. er effect heat pump which requires no heat exchanging radiators or chambers The only moving parts are cooling fans and a circulating pump Neither of these devices require maintenance and the cooling fans will run continuously whenever power is applied to the MSTC A removable cover on top of the enclosure provides access to the desiccant drying tube Access to the drying tube is required PROTEINSOLUTIONS www protein solutions com occasionally to change or regenerate the desiccant See Periodic Maintenance for instructions on changing the desiccant When power is applied to the TC MicroSampler the temperature controller does a quick self check and then displays two panels of numbers The value displayed in red is the current temperature of the cell chamber The lower value in green is the target temperature defined by the user The target temperature will be the last value entered from the previous session The default target temperature from the factory will be 20 Celsius When the temperature controller identifies a difference between the sampling chamber temperature and the target temperature it will either cool or heat the chamber until the values match It will continue to do this as long as power is applied to the instrument The target temperature can be set manually using the up and down arrow keys on the front of the TC MicroSampler or can be remotely controlled using the DYNAMICS V6 software The Event Schedule feat
56. ere standard are delivered facilitate direct syringe loading using a 2 inch needle thereby minimizing the possibility of contamination The recommended procedures for loading the sample syringe and filtering the Microsphere standard are as follows 1 Thoroughly clean the quartz cell with soap and water Rinse the cell with filtered DI water and then dry with compressed air 2 After attaching the needle to a 1 mL disposable syringe insert the needle through the dropper tip of the Microsphere container and extract approximately 0 75 mL of standard 3 Remove the needle from the dropper bottle tip the syringe upright needle up and pull any sample remaining in the needle into the syringe cavity PROTEINSOLUTIONS www protein solutions com 38 39 4 Place a 0 02 um Anotop filter in line between the syringe and the needle and then drip out 2 3 drops of sample into a waste container The dripping can be stopped by applying a slight backward pressure to the syringe plunger see Note below 5 Load the quartz cell by inserting the needle all the way to the bottom of the cell and dispensing sufficient sample to just fill the cell window Be careful not to scratch the cell window when placing the needle into the cell 0 02 um filter The cell is now ready to be loaded into the DynaPro Note that there is an opaque or frosted side on the cell The frosted side of the cell must be positioned to the left side of the optics block
57. ers MW R Model Globular Proteins Fined Cone mgimL 1 83 Instrument Solvent Vol mL 0 012 dnde mLig 0 187 UserDefined Tol Int Cntis 100000 E Measurements G Meas 1 7 E Meas 2 Click here to select H Meas 3 the measurement Notes amp Tips 1 If you routinely use the same parameter settings the File Save Settings option on the main menu bar can be used to save the current configuration including parameter values to the control file such that they will always be loaded when a new file is opened PROTEINSOLUTIONS www protein solutions com 77 Selecting a solvent Many of the calculations and data transforms in DYNAMICS V6 require solvent related information All of the properties and settings associated with the solvent are contained within the Solvent category of the Parameters Node Exp1 E e PS ie a E E Expt Click here to select solvent Hardware Property Value Parameters Fixed Instrument Name Parameter Source Rfr Idx 589nm amp 20C Sample Viscosity 20C cp UserDetined al Cauchy Coef nm Measurements Tal Temp Model F Meas 1 Baye ots PBS Standard 1333 1 019 3119 Aqueous Expl Hardware E Parameters Fixed Instrument Name Parameter Source Rfr ldx 589nm amp 20C Viscosity 20C cp Sample
58. g the following procedures PROTEINSOLUTIONS www protein solutions com 1 Center the mouse cursor over the data point and select the data point with a left click selected point will be highlighted in green 2 Open the Right Click Menu with a right click on the figure 3 Select the Mark option in the Right Click Menu Note that if multiple points have been selected the Mark option will change to Mark All dynamics MSX qa3 exp 101x amp Bere en EB Selected data noted D ae Ss S W2 1 by the green color _ ee Uli a MSX qa3 exp amp Hardware n a Mark Ctre amp Parameters E Measurements T Copy Ctrl C EB Meas 1 2400 Mark option in Trace 2 Data Filter Etrl F 5 Pp lick 2 Figure Settings View right click menu 22007 3 tao oe Control Panel 2000 Statistics 4 DynaGraph 18007 H 40 0 10 20 30 40 50 60 70 80 90 100110 Time s Note the absence of the Control Panel in this window removed 1992 003 Rimm by unchecking the option in the right click menu NUM Multiple data points can be selected by depressing the Ctrl key and then left clicking the additional points If multiple data points have been selected the Ctrl key can also be used to de select one or more points After Marking the outlying data point is removed from the Trace View display and is ignored in subsequent calculations such as the Regularization analysis
59. ght determinations If a titrator is present in the hardware configuration only the Conc mg mL value for measurement 1 is user definable all other Conc mg mL values are dependent upon the titrant injection volume i e the Inj Vol mL and are defined by the automatic titrator routines Note that if the user changes the concentration value for measurement 1 during or after the titration the concentration values for subsequent measurements will be updated accordingly di dC mL g The specific refractive index increment for the sample in units of mL g The value is used to calculate the Rayleigh ratio in static light scattering applications Ext Coef L mg The sample extinction coefficient in units of L mg The value is used along the UV absorbance to calculate the sample concentration in HPLC applications Inj Vol mL The milliliter volume of titrant injected prior to recording data for the designated measurement The parameter is only available if a titrator is present in the hardware configuration and it is used to calculate the subsequent sample concentration The value is determined by the automatic titrator algorithms and is not user editable PROTEINSOLUTIONS www protein solutions com 74 MW R Model The molecular weight vs radius model used to estimate the molecular weight from the hydrodynamic size of the analyte Available options are Globular Proteins Linear Polysaccharides Branched Polysaccharides and Sta
60. have questions regarding the analog output of your device or if you need a bias adapter to adjust the signal to a range suitable for use with the DynaPro RS232 COM Ports The RS232 ports are used to handle communications with the temperature module in the DynaPro optics block and other devices that require a communications protocol more advanced than simple analog digital I O Fiber Optic Connections The tongue and groove fiber optics ports are used for connecting the fiber optics from the DynaPro optics block The metal clad fiber optics cables carry the incident laser light from the diode laser in the host unit to the cell in the optics block and the scattered light from the cell back to the detector in the host unit PROTEINSOLUTIONS www protein solutions com 21 USB Connections The Universal Serial Bus USB ports are used for communications between the DynaPro host unit and the computer The USB ports on the back of the computer can also be used to establish communications with other USB devices supported by DYNAMICS V6 Power Connections The power cables are used to supply power to the DynaPro host unit from a standard wall receptacle The power out jack on the back of the temperature controlled optics unit is included for supplying power to the DynaPro host unit but can alternately be used for transferring power from the wall receptacle to other devices Military Jack The military jack is a special type of connection
61. he Statistics Table are added deleted and moved in a similar fashion The Format Data worksheet can also be accessed from the column right click menu Saving workspace settings If you routinely use a particular set of experimental parameters they can be saved to the control file using the File Save Settings command on the menu bar When this command is used all of the settings for the current selected experiment are saved including hardware configuration table and figure settings all properties and values from the Parameter Node Event Schedule commands and application wide options and will be automatically loaded when the next new experiment is opened Additionally workspace settings and values can also be save to a user defined PreSet file using the Save As PreSet command PreSet files can be opened and re used at later dates via the Open PreSet option on the menu bar Click here to save current experiment settings as deault settings for next New experiment DYNAMICS Exp File View Tools Window Help New Open Close Save i litud Open PreSet tem Time Temperature Amplitude Save As PreSet s Print Preview Click here to save the Print Setup experiment settings to a 199 270 gal V6 exp user defined PreSet file 2 CAWINDOWS BSA 6 26 exp Exit For Help press F1 PROTEINSOLUTIONS www protein solutions com 93 Defining solvents in DYNAMICS V6 Solvents are defined
62. he measured correlation curve has returned to the baseline within the time encompassed by the defined number of channels Deviations from the theoretical value of 1 000 indicate either a noisy baseline or a range of correlator channels that is too small Conc Al The sample concentration determined from an analog detector connected to the BNC jack If the detector is an RI detector the Conc Al value is determined directly from the Conc Al vs RI AI calibration curve If the detector is a UV detector the Conc AI value is determined using the Abs Al vs UV AI calibration curve the value for the Abs AI Baseline and the Ext Coef value D The apparent analyte diffusion coefficient determined using a Cumulants analysis Diam The apparent diameter of the analyte determined using a Cumulants analysis H Al The raw analog data from a pH detector connected to the BNC jack Intensity The measured scattering intensity of the sample KC R90 The Y axis value in a Rayleigh plot of KC R90 vs concentration where the intercept is equivalent to the inverse of the molecular weight and the slope proportional to the second virial coefficient PROTEINSOLUTIONS www protein solutions com 75 KC R90 Al The Y axis value in a Rayleigh plot of KC R90 vs concentration where the intercept is equivalent to the inverse of the molecular weight and the slope proportional to the second virial coefficient The AI component indicates that the sam
63. he needle tip upwards as the sample is dispensed 2 While not pictured above a 100 uL pipetman with capillary tips also works well for loading cells especially if you choose to centrifuge rather than filter your sample If you use a pipetman it s recommended that you blow any dust out of the tip with compressed air prior to filling with sample 3 The bowl at the top of the cell window tends to collect dust It is recommended that you place only enough sample in the cell to fill the window Sample Preparation Troubleshooting When I load the cell I still occasionally get a bubble even when pulling the needle tip upwards during loading This is a common problem with low volume cells Try tipping the needle into one of the comers of the cell window A lot of times that will pop the bubble If you have sufficient sample volume you might alternately try using a larger volume cell The 45 uL cell tends to be much easier to work with when it comes to avoiding bubbles PROTEINSOLUTIONS www protein solutions com 36 THE MICROSPHERE STANDARD PROTEINSOLUTIONS www protein solutions com What is a Microsphere standard The Microsphere standard is a proprietary formulation of polymer spheres with known scattering intensity and hydrodynamic size The standard is stable at ambient temperatures for up to two years and is suitable for filtration through 0 02 um Anotop filters Protein Solutions uses this standard for both Quali
64. ide of the Experiment Window and then select the host and optics units for your DynaPro from the Host Serial Number and Optics Serial Number list boxes in the properties table If you have a temperature controlled optics block you ll also need to select the TC Unit option in one of the Serial COM list boxes it doesn t matter which COM port you select the software will find the correct one when the Connect button is clicked Note that if you have only 1 host and 1 optics block DYNAMICS V6 will automatically load those devices into the Hardware Node The Host and Optics Serial Number list boxes are included for the increasing number of PSI clients with multiple DynaPro systems PROTEINSOLUTIONS www protein solutions com Open new file here Edit View Window Help S E Se Select DynaPro host and optics units here Property Host Serial Number Optics Serial Number Analog Input 1 empty Analog Input 2 empty Analog Input 3 Serial COM 1 Serial COM 2 Parameters Event Schedule Measurements Connection Status Not Connected Connect Disconnect Connect to DynaPro 4 host unit here For Help press F1 NUM Z Once the hardware configuration is defined insure that the power is turned on and then click the Connect button at the bottom of the window After a few moments the Connection Status text box should report that
65. igned to handle 0 to 2 5 V analog signals Negative signals can damage the chip Prior to connecting an external detector please insure that the specified output signal for the detector is within the acceptable specifications 0 2 5 Volts for the DynaPro Connecting the USB and power cables WARNING When the DynaPro is connected to the computer via a USB cable Windows will automatically detect new hardware even if the DynaPro is un powered If DYNAMICS V6 has not installed the correct USB drivers Windows may install the wrong ones While DYNAMICS V6 can recovery and correct the problem it is a multi step process that can be avoided if the software is installed prior to connecting the USB cables Communications between the computer and the DynaPro are handled through a USB port on the back of the host unit Depending upon the model some DynaPro host units have two USB ports If your host unit has two USB ports both should be connected to USB ports on the back of the computer Note that it is irrelevant which USB cable is connected to which port for multi USB port instruments Once the USB connections are completed the power cable from the host unit can be connected to a standard wall receptacle PROTEINSOLUTIONS www protein solutions com 25 Establishing Windows DynaPro USB communication links To insure that all USB drivers are properly installed turn the DynaPro power Once the powered instrument is connected
66. ime h a lonic strength M Reactor temp C DO C 2 Use the Add button to add Be the parameter to the table loric stength M SS Remove PROTEINSOLUTIONS www protein solutions com 79 To add a User Defined parameter to the property table select the measurement from the Measurement list box select the parameter from the Parameter list box and then click the Add button The parameter along with its defined units will be added to the property table and the value for the parameter can then be entered directly into the value cell double click to edit User Defined parameters are deleted from the properties table by selecting the row containing the parameter and then clicking the Remove button in the panel below the table Notes amp Tips 1 User Defined parameters along with their units are defined and added to the database in the Tools Parameters User Defined window accessed from the main menu bar 2 User Defined parameters can also be displayed in the Grid and Trace Views If the defined units are supported by DYNAMICS V6 they can be transformed in the Grid View by right clicking the Units cell Monitoring data with the Instrument Control Panel Prior to starting an experiment it is generally a good idea to check the quality of the data using the Instrument Control Panel The DYNAMICS V6 Data Monitor function is automatically launched when the Instrument Control Panel is opened using the Dy
67. ings for hardware components can be edited in the properties table for the component To edit a property value double click the Value cell text will highlight in blue and then enter the new value Specific properties for A D and RS232 devices are discussed in the Application Oriented Software tutorial Expl 15 x E e e ei j des Serial Number Optics test Model Titrator J Internal Laser Parameters Measurements Laser Wavelength nm Analog Digital WO Exp1 Expl 3 Hardware Property Host test Titration Type aiins test Sample Syringe Vol mL ite Titrant Syringe Vol mL Cell Type Flow Syringe Speed s stroke 5 wu Max Sample Vol mL Parameters Measurements PROTEINSOLUTIONS www protein solutions com 70 DYNAMICS V6 parameters The following is a list of the reserved use parameters in the DYNAMICS V6 software Hardware Node Analog Input Distinguishes the BNC jack used for an analog signal input from an external detector Auxiliary HW Indicates the presence of auxiliary hardware with properties that have been integrated into the DYNAMICS V6 algorithms e g Auxiliary HW 1 HPLC Pump with a Flow Rate mL min that is used along with the Offset Vol mL for a UV detector to align scattering intensity and absorbance peaks in a chromatogram
68. ior to connecting the instrument If the DynaPro is connected to the computer prior to the installation of the software the Add New Hardware Wizard in Windows will not be able to find the compressed drivers Should you find yourself in this situation use the Browse button in the Add New Hardware screen and point the routine to the DynaPro Drivers and QC Files floppy disk delivered with your instrument If you encounter any problems with driver installation contact technical support at 1 888 242 2848 If you are using the disk version of the tutorial then DYNAMICS V6 has already been installed and you can move on to the Unpacking the DynaPro section of the tutorial If you are using the hardcopy version of the Setup tutorial and are setting up the DynaPro for the first time then you should install the software now The procedures for installing the DYNAMICS V6 software are as follows 1 Insert the installation disk into the CD Rom drive If the properties of the drive are set to Auto start the DYNAMICS V6 Setup package will start automatically Alternately you can use the Start Run command on the Windows Task Bar Use the Browse button in the Run window to find the Setup exe file on the installation CD 2 After installing a few temporary files the DYNAMICS V6 Setup package will ask for the Application Path The recommended path is c Program Files Protein Solutions While you can select an alternate path using the Browse button instal
69. is ready to begin recording While the incoming data stream can be monitored from the Instrument Control Panel none of the data is being saved to memory when the system is in the monitor mode Data recording is started by clicking the green Record button in the Experiment Window The Record button face will change to a flashing red indicating that DYNAMICS V6 is recording data and incoming data will be displayed in the Grid View or whichever view is selected on the tool bar of the Experiment Window Data recording is stopped by clicking the flashing red Record button The Record button face will then change to green indicating that software is ready to begin recording data for the next measurement PROTEINSOLUTIONS www protein solutions com 81 Record Button in Recorded acquisition data the Recording Mode for measurement 4 one acq per me s exp Em wl El one acq per meas exp Hardware Intensity Absorbance Parameters Cntis Measurements 400000 175000 350000 250000 250000 350000 476000 Meas 1 Meas 2 14 1 191 24 1 29 1 344 Avg 24 1 277778 25 19 1 6333 0 s 12 9 93871 0 5623 0 0709 0 EEE 1 7500 1 5400 4 7225 Notes amp Tips 1 2 3 While the Experiment Window is in the recording mode incoming data is recorded regardless of the vie
70. is un coded and needs to be connected to the APD detector The fiber optics can only guide light properly when they are connected correctly This is VERY important Please carefully inspect and follow the alignment procedure given below PROTEINSOLUTIONS www protein solutions com 22 23 Insert the fiber optic into the fiber optic port on host unit Align the raised notch on the optic cable with the groove in the fiber optic port Push inward gently until the notch is seated in the groove Thread the sleeve onto the fiber optic port until no threads are visible DYNAMICS V6 communicates with the optics block temperature sensor and stir pad through a military cable connection between the host unit and the optics block The military cable is permanently fixed to the back of the optics block Note that there are two components to the military plug the jack and the locking sleeve To connect the military plug hold the plug by the jack component insert the plug into the receptacle and turn while pushing slightly inward until you feel the plug seat itself in the correct position Then push the sleeve inward and turn clockwise as far as possible PROTEINSOLUTIONS www protein solutions com Connecting RS232 cables DYNAMICS V6 communicates with the temperature control module in the optics block and other supported COM port devices e g automatic Titrator and Ultrospec 4000 via RS232 serial COM port cables To e
71. istinguished by the presence of a single needle port in the lid on the top and a variable speed control knob on the back of the optics unit TC Upgrade True or False indicator for whether or not the optics unit includes temperature control functionality PROTEINSOLUTIONS www protein solutions com 71 A D Detector and Auxiliary Device Nodes Calibration The set of data points describing the analog input mV dependence of the absorbance UV detectors concentration RI detectors or pH meter or probe for an external analog detector Calibration data is accessed and or edited using the Show button in the value cell Cell Pathlength cm Distance light must travel to pass through the sample or the length normalization factor b in Beer s law Abs ebC The Cell Pathlength cm is used along with Ext Coef L mg to calculate the sample concentration from the UV absorbance This parameter is only available if the A D detector is of the UV detector type Detector Type Indicates the type of analog detector Current options are pH UV and RI differential refractive index The Detector Type value determines the availability of other A D detector properties e g if the detector is a UV detector the Cell Pathlength nm and Ext Coef L mg properties will be added to the Hardware and Sample Nodes Model amp Serial Number Used to identify A D detectors and auxiliary devices Offset Volume mL The cell to cell displa
72. ithout the permission of Protein Solutions This warranty does not include operating supplies The customer is responsible for replacing common disposable parts as may be necessary without on site assistance from Protein Solutions It should be noted that with the exception of the desiccant in temperature controlled MicroSamplers there are no user serviceable parts contained within this Instrument Any signs of tampering with the instrument will invalidate this warranty If this Instrument is on lease from Protein Solutions the warranty coverage is extended over the lease period agreed by both parties with all no tamper rules applying Any cost s involved in the repair of instruments considered to have been tampered with will be the responsibility of the purchaser or lessee Statement Of Liability Protein Solutions endeavors to ensure that the information in this document is correct and fairly stated but does not accept any liability for any error or omission The development of Protein Solutions products and services is continuous and published information may not be up to date Any particular issue of a product may contain only part of the facilities described in this manual or may contain facilities not described herein If there are any questions please contact Protein Solutions Specifications and statements in this manual as to a product s performance are Protein Solutions estimates and are intended f
73. k only when the solenoid is triggered by the humidity control circuit A pressure regulating valve on the IN side limits gas pressure However a maximum gas pressure of bar should be observed When operating at low temperatures over extended periods ice may form on surfaces within the enclosure This is unavoidable and is caused when the enclosure is open to the atmosphere i e if the door is left open where moisture in the air condenses and freezes very quickly on the cold surfaces and no change in humidity is detected If this occurs the system should be heated to disperse the ice before switching the instrument off This procedure reduces the risk of unwanted water drips entering the rest of the enclosure Important Note If your DynaPro is fitted with remote air drying connections the red end plugs must be fitted when operating with the self contained closed loop drying system Periodic Maintenance The MicroSampler is very easy to maintain For trouble free operation during your ownership period follow these simple guidelines Cuvettes If the clean water count rate from the cuvette appears to be higher than normal after repeatedly flushing with water clean the cuvette with a mild detergent solution and then rinse thoroughly with DI water until the correct clean water count rate is reached The cuvettes can be stored in the small trough under the access lid of the TC when they are not in use or in the boxes supplied Out
74. l from the molecules being measured The total sample injection volume including filtration is approximately 20 uL if the 12 uL cell is used or 55 uL if the 45 uL cell is used After the cell is placed in the MicroSampler the sample is illuminated by the laser The scattered light is correlated in the DynaPro host unit which then sends the results to the PC for analysis by the DYNAMICS V6 software package Incident laser light 90 scattered light Other detectors PC with DV6 for data analysis Setting Up The Instrument There are three cables between the MicroSampler and the DynaPro host unit 2 fiber optic cables and a military cable electrical signal leads encased in stainless steel The cables are permanently attached to the rear of the MicroSampler and need to be connected by the user to the rear of the DynaPro in the appropriate sockets Take great care with the optic signal leads and insure that the keys on the cables are aligned with notches in the connectors before screwing down the locking caps PROTEINSOLUTIONS www protein solutions com RS232 Cable Optional Temperature Control External External RS232 Analog Digital Devices Devices BNC Cables Military Cable Fiber Optic RS232 Cables USB Cable DynaPro Host Unit Diode laser Launch Optic APD Receiver Optic Correlator Cell Chamber 24 bit A D VO MicroSampler 12V power supply Co
75. lation of future upgrades and add ons are simplified if you use the recommended path 3 After creating the appropriate directories the DYNAMICS V6 Setup package will present the Installation Options window If the Custom Setup is selected you will then have the opportunity to select which components you wish to have installed to the hard drive Note that options not installed at this time can be installed from the same window at a later date by repeating the installation process 4 When the installation process is completed the computer should be restarted to insure that all files are recognized by the Windows operating system PROTEINSOLUTIONS www protein solutions com 15 DYNAMICS V6 Installation Options If the Custom option is selected during the software installation the user can choose which components of the software are to be installed Descriptions of the optional components of the DYNAMICS V6 software package are given below Select Components Eg Select the components you want to install clear the components you do not want to install Components MY Dynamics V6 iv Manuals 8039 K M PSI Books 19157 K WV Support Utilities 6080 K Destination Directory C Program Files Protein Solutions Browse Space Required 45324 K Space Available 2096832 K Disk Space Som Trstallotrela DYNAMICS V6 This is the instrument control data collection and data analysis compone
76. lected parameter i e value displayed true value x scalar Notes amp Tips 1 Figures can be copied to the Windows clipboard using the Copy command in the right click menu To copy a figure into a Word document right click the figure select Copy from the popup menu select a location in your manuscript and then select Edit Paste from the Word menu bar or use the Ctrl V shortcut The figure will be pasted into the Word document as a bitmap image Displaying the size distribution The size distribution for the measurement Meas or acquisition Acq selected in the Tree View can be displayed using the Regularization View button on the Experiment Window tool bar PROTEINSOLUTIONS www protein solutions com 54 ilh Display Regularization View dynamics MSX qa3 exp iof x fi Eile Edit View Window Help 18 x Deu els mle E MSX qa3 exp m Control Panel E Hardware A may cio Parameters Size distribution for erage 1 El Measurements Measurement 1 eg Meas 1 D 20 5 Optimal Right click menu 10 x Anis 1 Radius x Copy Ctrl C Ott e ttt Y Axis 0 01 0 10 1 00 10 00 100 00 1 00E 03 1 00E 04 intensity Data Filter Ctrl F Radius nm Zintensity Figure Settings Model v Control Panel Spheres Aiea Item R Pd lntensity Mass TE umves DynaGraph nm MPeak1 08 12 21 54 T Legend
77. low Kit Upgrade UttroSpec 4000 False Titrator True Min Lys Cone mg mL 0 1 Toluene Int Cnt s 100000 Water Int Cntis 10000 Remove A D Detectors C Auxiliary Devices PROTEINSOLUTIONS www protein solutions com When the Original Equipment radio button is selected the properties and values necessary to completely define the original equipment delivered to the end user are presented in the properties table As of February 2001 the parameters and values defining the original DynaPro equipment are programmed into the instrument The information is also printed to a label on the inside cover of the software installation disk contact support protein solutions com if you cannot find the original equipment information Original equipment properties and their use in DYNAMICS V6 are as follows Host Serial Number Defines the specific DynaPro host unit Host Model Defines the model of the DynaPro host unit Currently there are 2 types of host unit models 99D and 99E 99E models have a single USB PC connection while 99D models have two USB PC connections IFlex Laser Identifies whether or not the DynaPro diode laser is contained within the host unit IFlex True or within the optics block IFlex False Host units with IFlex lasers have two fiber optic connections on the back of the host unit one for the launch optics and one for the avalanche photodiode detector Optics Serial Number Defines the specific Dyna
78. lumn Click and drag to in selected view or format the data move a column Table Settings eres Font Size fia x tem Time Intensity Amplitude D R Pd Baseline SOS Cntis Remove Column Format Data Select view type here Options Statistics Rows Edit Options View Options Temperature Add Grid View Pd Index C Regularization View Add All Sa Statistics Table Remove All cen Column Row options for selected view az Statistics Table rows PROTEINSOLUTIONS www protein solutions com 92 The table to be formatted is defined by the selection in the View Options frame on the Table Settings worksheet For the tables in the Grid and Regularization views the user can define 1 the columns displayed 2 the order of the columns 3 the column units and 4 the format general fixed or scientific of the column data For the Statistics Table the user can define the rows or statistics to be displayed and the order of the rows in the table The available column row headers in the Options list box vary with the View Option selection Columns are added to the table by selecting a column header from the Options list box and clicking the Add button Columns are removed using the Remove Column option in the right click menu right click on the column header to open the menu Columns are moved by left clicking and dragging Rows in t
79. mand will copy the figure or selected data in the Results Summary to the Windows clipboard for Pasting into other Windows applications The Export command will copy the data for the figure or the selected data in the Results Summary table to a user defined file in text format Results Summary The Results Summary table gives a per peak breakdown of the size distribution including the mean size polydispersity standard deviation of the peak and lntensity and Mass contributions to the signal The table and columns can be formatted to the user s specifications by selecting the Table Settings option in the right click menu The check boxes in the first column are included to facilitate removal of a peak from consideration in the lIntensity and Mass calculations This feature is particularly helpful for very low concentration samples where noise in the correlation curve at long delay times can lead to the erroneous appearance of small peaks at large sizes gt gt 1 micron well outside the range of dynamic light scattering instrumentation Resolution Slider The peak resolution limit in dynamic light scattering is 2x in size or roughly 5x in molecular weight In other words if your system is composed of a monomer PROTEINSOLUTIONS www protein solutions com 91 and a pentamer it is likely that the two particles can be resolved with dynamic light scattering measurements whereas a smaller molecular weight range is very unlikely to
80. mputer with DYNAMICS V6 Upgrade Options Temperature Control Flow Mode Ports Build In Stir Pad If you have a temperature controlled TC MicroSampler you will need to use the included RS232 cable to connect the MicroSampler to a COM port on the PC Additionally there may be optional connections on the rear of the TC instrument for external fluid cooling if this has been requested prior to purchase See Temperature Controlled MicroSamplers for details on the use of the fluid cooling connections If your DynaPro instrument includes a Flow Kit you will also have analog and digital BNC connection jacks on the rear of the DynaPro host unit for connecting external devices and detectors Using The DynaPro Access to the sample chamber is gained by lifting the front lid on the MicroSampler The sample cell low volume quartz cuvette is inserted into the aperture and the lid closed for measurements Note that there is a safety switch connected to the MicroSampler lid that will disconnect the laser when the lid is open Prior to recording light scattering data you must first establish communications between the DynaPro and DYNAMICS V6 by clicking the Connect button below the properties table of the Hardware Node in the Tree View select Hardware in the Tree View and the Connect button will appear beneath the table on the right side of the window Once the instrument is connected you can begin recording data by click the green Record button on the
81. n filtering many DynaPro users prefer to centrifuge their samples As a consequence of the centripetal force larger dust particles migrate to the bottom of the centrifuge tube thereby eliminating the need to remove the dust particles via standard filtration techniques Recommended spin rates and times vary with the sample However 10 15 minutes at 3000 RPM is typical When removing the sample for cell loading remember that only the top portion of the sample is dust free Notes amp Tips 1 Because of the volume control a Pipetman works best for transferring the sample from the centrifuge tube to the DynaPro cell Dust can be removed from the pipette tip with compressed air Loading the cell Sample is loaded into the DynaPro cell by placing the needle or pipette tip all the way to the bottom of the cell and dispensing the specified volume of sample Be careful not to scratch the cell window when placing the needle into the cell When loading the cell into the MicroSampler you should note that there is an opaque or frosted side on the cell The frosted side of the cell must be positioned to the left side of the optics block After seating the cell in the DynaPro optics block close the lid to de activate the laser power safety kill switch You are now ready to begin collecting light scattering data PROTEINSOLUTIONS www protein solutions com 35 Notes amp Tips 1 If you encounter bubble problems try slowly pulling t
82. naPro icon on the Experiment Window tool bar In addition to the Data Monitor function the stir pad acquisition time and laser power can be controlled from the Instrument Control Panel Instantaneous DynaPro and external detector readings Time s Intensity Cnt s _ Temperature g Waters RI mY Absorbance Control stir pad here av Instrument Contol Panel m Stir Cell 1 654892 24 8 Start Stop 2 685391 24 8 3 666827 24 7 4 R772905 24 8 m Settings Acquision Time E 4 Adjust the laser nny power here 100 y bb4374 T Auto Adjust Recommended number of acquisitions for current sample m Correlation Noise Factor Sol Ent s CNF 0 0285 30589 Optimal Acq 12 Ty Use water Check here to use the clean water count for your DynaPro rather than the solvent intensity to calculate the Correlation Noise Factor PROTEINSOLUTIONS www protein solutions com 80 The Correlation Noise Factor CNF is indicative of the noise in the dynamic light scattering correlation curve for a given sample acquisition time The CNF is calculated from the sample and solvent count rates The Optimal Acq value in the Instrument Control Panel is derived from the CNF for a single correlation curve and represents the expected number of acquisitions that need to be collected in order to achieve an acceptable signal to noise ratio
83. nd Y Axis list boxes in the Control Panel The Y Axis list box utilizes a check box format allowing multiple selections There are currently no limits with regard to the number of parameters that can be displayed on a single figure The units for the Intensity Cnt s shown on the left axis and represented by the blue line and the Radius nm shown on the right axis and represented by the red open circles are the same as the units defined in the PROTEINSOLUTIONS www protein solutions com 84 Grid View For display purposes a 0 001 multiplier was applied to the Intensity The multiplier was defined using the Scalar text box in the Control Panel and is noted for quick reference in the Legend The Legend can be hidden by un checking the appropriate box in the Control Panel kr Display Trace View dynamics fy Eile Edit Window Help DERW ts e S e 1 m Select the parameters v Eli a 7 to be displayed here E MSX qa3 exp ae Control Panel E Hardware X Axis ZN E Parameters a Time be E Measurements T DE m 5 YAris Click here to change s 56 from line to symbol E E Mintensity 22007 a Temperature 2 1 5 0 3 Amplitude 2 2000 oD Apply a scalar to the E 45 MR fd selected data set here 1800 V Line Tt tt tt 444 ae Scalar 0 001 2 0 10 20 30 40 50 60 70 80 90 100110 Left Right er the display i ax s nere Time s Legend Jo
84. ndependent of the model If multiple selections have been made in the Tree View the View list box can be used to convert the figure to a 3 dimensional bar graph Descriptions of the options available in the Regularization View are given below ah Display Regularization View dynamics MSX qa3 exp File Edit View Window Help peure E MSX qa3 exp Display E Hardware Options Parameters MM E E Measurements Radius x a 2 Decay Time 5 Optimal Diameter n Diff Coef Right click menu 3 A Radius i Radius x C Clee Aue intensity Export 0 01 0 10 1 00 10 00 100 00 1 00E 03 1 00E 04 lntensity x Mass Data Filter Ctrl F Radius nm Kiauresetings Model Spheres x Spheres z Coils v Control Panel View p o ity ST Item Pd lntensity Mass cuves am Hyran MPeak1 08 12 21 54 T Legend Cuves A Peak2 62 23 79 46 NEE Bars Results Summary For Help press F1 FORUM Right Click Menu The Regularization View right click menu contains the standard functions and options available in most of the DYNAMICS V6 right click menus The Control Panel and Results Summary can be hidden by deselecting the appropriate option in the menu The Data Filter along with the Figure or Table Settings windows depending upon where the menu is activated can also be opened from the right click menu The Copy com
85. ng data results from other detectors UV refractive index etc and apriori information supplied by the research scientist 3 Not only collect and store data but assist with the interpretation 4 Perform common time consuming laboratory tasks such as designing buffer recipes converting units and calculating concentrations 5 Free the user for other tasks by automating routine and user defined experiments 6 Evolve with the needs of the user 7 Remember 8 Teach All of the above and more That s what DYNAMICS V6 was designed to be What does data mean In DYNAMICS V6 data is defined as any piece of information that the software can use to assist the researcher in their sample characterization studies Within DYNAMICS V6 data is divided into 3 classifications user defined calculated and time dependent Time dependent data is further divided into 2 categories instantaneous readings collected every second and PROTEINSOLUTIONS www protein solutions com 59 60 acquisitions average of readings collected across a user defined acquisition time Along with the time average of the instantaneous readings an acquisition also defines a correlation curve i e the intensity correlation data for a single dynamic light scattering measurement All the above types of data can be stored displayed compared and utilized for further calculations and or search routines within DYNAMICS V6 When viewing data in DYN
86. ng the symbol Hardware components are added or removed using the dropdown list boxes in the properties table at the Hardware Node Should you have PROTEINSOLUTIONS www protein solutions com 69 multiple DynaPro systems you can use the Host and Optics Serial Number properties to distinguish the systems Record Button disabled until all hardware is connected Value empty Host Serial Number 99 D 50 Waters UV TER DS Waters Rl Optics Serial Number BNC pH probe Analog Input 1 empty Analog Input 2 empty Analog Input 3 empty Serial COM 1 empty Serial COM 2 empty Current list of user defined analog detectors Property Host 99 D 50 Optics LSA 045 Parameters Measurements Supported RS232 devices empty Connection Status Other Devices UltroSpec 4000 Not Connected a Connect Titrator za AutoSampler C USE Disconnect z Aux The Analog Input list boxes contain a listing of all analog detectors defined by the user in past experiments The Serial COM list boxes contain a listing of DYNAMICS V6 RS232 supported devices currently available to the user As hardware components are added and or removed the expanded Hardware Node in the Tree View is updated to reflect the new hardware configuration The Connect and Disconnect command buttons are used to open and close communications with the current hardware The Add button in the Other Devices fr
87. ng the cell Sample Preparation Troubleshooting PROTEINSOLUTIONS www protein solutions com WO ONNNNANADA AD 15 15 16 17 18 18 20 22 24 24 25 26 26 21 28 29 30 32 32 33 33 35 35 36 The Microsphere Standard What is a Microsphere standard Preparing the Microsphere standard Loading the Microsphere PreSet Measuring the Microsphere standard Interpreting the Microsphere results Microsphere Troubleshooting Simple Batch Experiments Simple Batch Experiments Overview Preparing the sample Checking the hardware configuration Batch experiment parameters Collecting data Setting up the Grid View Preparing a figure Displaying the size distribution Printing a one page summary Simple Batch Experiments Troubleshooting Using DYNAMICS V6 Using DYNAMICS V6 Overview What is DYNAMICS V6 What does data mean Launching DYNAMICS V6 Structure of DYNAMICS V6 Opening a new experiment file Data grouping in the Tree View The Experiment Window Tool Bar Defining hardware Defining the hardware configuration Setting hardware properties DYNAMICS V6 parameters Setting experimental parameters Selecting a solvent User defined experimental parameters Monitoring data with the Instrument Control Panel Recording data Displaying data in the Grid View Graphical displays in the Trace View Marking outlying data points Using the Data Filter Real time data filtering A note on saving Marked data The Co
88. nt of the DYNAMICS V6 software suite The DynaPro cannot be operated without this component Manuals Select this option if you wish to have the DynaPro User Manual and the QuickStart Manual installed to the hard drive The User s Manual is an MS Word document containing all of the information text and figures from the DYNAMICS V6 Help file The QuickStart is a PDF document identical to the hardcopy QuickStart manual delivered with the DynaPro Molecular Sizing Instrument Both documents are formatted for printing PSI Books PSI Books is a library of articles in Help File format designed to assist DynaPro users in data interpretation In contrast to a simple help document which addresses how things happen PSI Books utilizes a teaching approach and addresses not only how but why things happen References and Application Notes are also included in PSI Books Support Utilities The Support Utilities component of the DYNAMICS V6 software suite contains a variety of helpful Excel spreadsheets PSI written software utilities and 3 party public domain software The content of the Support Utilities package evolves with time depending upon which utility functions have been fully integrated into DYNAMICS V6 PROTEINSOLUTIONS www protein solutions com Unpacking the DynaPro The DynaPro instrument was delivered in two heavy duty cardboard boxes with foam inserts To minimize free movement of the instrument during shipmen
89. o add a column to the Grid View insure that Grid View is selected in the View Options frame select the column header to be added from the Options list box and then click the Add button in the Edit Options frame To change the column order left click the column header and drag it to the desired location The column header and units menus are also active in the Table Settings worksheet To access the popup menus right the appropriate cell in the Columns frame Column Row options for selected view There are 3 types of tables in DYNAMICS V6 the Grid View data table the Results Summary table in the Regularization View and the Statistics Table The View Options frame in the Table Settings worksheet allows the user to format all three of these tables at one time To setup the columns in the Results Summary table in the Regularization View select the Regularization View option To setup the rows in the Statistics Table select the Statistics Table option The column and row options available in the Options list box will change to be consistent with the selected view option Preparing a figure The Trace View gives a graphical presentation of the data in the Grid View The display can be changed to the Trace View by clicking on the Trace View button on the Experiment PROTEINSOLUTIONS www protein solutions com 53 Window tool bar The figure in the Trace View can be displayed in a multitude of user defined formats via the options avail
90. ode of the Tree View Parameters Node This node contains all user defined parameters and settings necessary for describing the experimental conditions e g pH TX100 concentration Lot etc and all parameters and settings essential for calculations e g solvent viscosity for calculating the radius Parameters and settings are added removed and edited by selecting the appropriate sub category in the Parameters Node Event Schedule This node contains a schedule of the user defined actions or events that are to occur or did occur during the course of an automated experiment There are no sub categories associated with the Event Schedule Measurements This node contains all the measured and calculated data collected during the course of an experiment Sub categories in this node are the individual measurements each of which is further broken down into Acquisitions Acq and Readings Read The PROTEINSOLUTIONS www protein solutions com 63 display format for the information in the Measurement Node is dependent upon which view button is selected in the Experiment Window tool bar The Experiment Window tool bar The buttons on the tool bar in the Experiment Window are used to select the display format of the data contained in the Measurement Node of the Tree View to start and stop data recording and automated experiments and to open various worksheets and control panels Brief descriptions of each button are given bel
91. olecular Sizing System The MicroSampler is the static and dynamic light scattering module for the system and contains the cell chamber and the focusing lenses for the fiber optics If your instrument includes a temperature control upgrade the temperature control components are contained within the MicroSampler With this type of system the modules are most appropriately stacked with the host unit on top of the MicroSampler A If your instrument performs measurements under ambient temperature only then the MicroSampler is best stacked on top of the host unit B B When initially stacking the modules provide room to access the back of the instruments for the USB cable power cables serial cable and fiber optics connections Do not switch either instrument on before all connections are securely established The detector is extremely sensitive and can be damaged beyond repair if exposed to room light Titration amp HPLC setup considerations If a Flow Kit upgrade was included with your DynaPro then your shipping list should include a low volume quartz flow cell The flow cell is interchangeable with the standard batch mode cell delivered with all DynaPro units and adds versatility to the instrument by facilitating flow mode experiments such as automatic titrations and HPLC However these types of applications do require special considerations during instrument setup The flow cell is seated in the optics block with the arrow on th
92. on holes on the front sides and top of the instrument are vital to the cooling performance in temperature controlled MicroSamplers and should not be obstructed at any time e Ifthe MicroSampler is fitted with fluid cooling heating enhancements the optional tube fittings carrying water are tested to local mains water pressure but will easily exceed this limit e On earlier models a 25 way D connector labeled DATA ACQUISITION ONLY carries electrical signals and DC supply voltages DO NOT under any circumstances attempt to connect a computer or other device to this port Permanent damage will occur e Some electrical components within the instrument will only operate at specific mains voltages In the unlikely event that your DynaPro is relocated to a country with a different mains voltage supply some internal components will need to be exchanged Call Protein Solutions if this occurs e When disconnecting the external coolant supply ensure the TC power is disconnected and the coolant supply pressure is reduced to zero Otherwise there is a danger of splashing coolant over the unit and causing damage e It is recommended that any time a problem arises you should turn off the instrument immediately to avoid any possible damage and refer to this manual or to your Protein Solutions Technical Representative to identify the problem and subsequently resolve it Some errors can cause damage to the internal components if the user
93. or general guidance only They may require adjustment in particular circumstances and are therefore not formal offers or undertakings Samples and example data in this manual are for illustrative purposes only The product described in this manual includes technology developed by the UK Ministry of Defense Royal Signals and Radar Establishment and licensed to Protein Solutions by Defense Technology Enterprises Limited PROTEINSOLUTIONS www protein solutions com Validity Of This Document This manual assumes the use of the DynaPro with DYNAMICS software and refers to this configuration in all diagrams and descriptions Please note that this manual is valid for all models Technical Manual Overview The DynaPro Dynamic Light Scattering Instrument is straightforward to operate so that knowledge of the underlying physics of molecular sizing is not essential The DynaPro Technical Manual is included purely to assist the interested user in understanding how the DynaPro produces its results For those interested in the mathematics underlying the calculations performed by the DynaPro the Theory of Operation section provides a more technical discussion of the techniques employed DynaPro Block Diagram The block diagram below summarizes the basic operation of the DynaPro Solutions are injected into quartz cuvettes and placed into the optics area manually The sample is filtered to eliminate dust particles which might interfere with the signa
94. ow Specific details are given in the appropriate sections of the tutorial Correlation View Record Button Wizard Checklist Titrator Control Panel Trace View Regularization View Grid View Displays the data and parameter values associated with the selection in the Measurement Node in a table format Other than direct data editing the features and available functions in the Grid View are similar to those incorporated into standard spreadsheet type software packages A statistical analysis of the data is also available in the Grid View via the right click menu All data including that contained in the Parameters Node can be displayed in the Trace View History Monitor Instrument Control Panel Trace View Displays the data and parameter values associated with the selection in the Measurement Node in a figure or graphical format Format and display features are similar to those in standard graphing software packages with the added benefit of having the displayed data linked to application specific algorithms and worksheets All data including user defined parameters can be displayed in the Trace View Correlation View Displays the correlation curve associated with the selection in the Measurement Node Overlay and complementary view options include best fit curves calculated and theoretical baselines channel cutoffs and residuals Regularization View Displays the size distribution derived from a Regul
95. ple concentration is derived from an analog detector connected to the BNC jack MW AI The absolute molecular weight of the analyte in the limit of infinite dilution where the second virial coefficient term is negligible The MW AI value is determined using the sample concentration derived from an analog detector connected to the BNC jack and is equivalent to the inverse of the KC R90 Al value Pd The polydispersity of the sample determined using a Cumulants analysis Pd Index The polydispersity index of the sample determined using a Cumulants analysis pH Al The sample pH determined from the pH Al vs H Al calibration curve for an analog pH detector connected to the BNC jack R The apparent sample radius determined using a Cumulants analysis R90 The residual Rayleigh ratio of the sample R90 Al The residual Rayleigh ratio of the sample using the concentration determined from an analog detector connected to the BNC jack RI Al The raw data from an analog RI detector connected to the BNC jack SOS The error or sum of squares difference between the measured and Cumulants calculated autocorrelation curves Temp The temperature measured by the sensor in the optics block Time The elapsed time from the start of the measurement UV AI The raw analog data from a UV detector connected to the BNC jack Setting experimental parameters Experimental parameters are defined at the Parameters Node which can
96. ratching the surface of the cell The cell can then be placed into the MicroSampler insuring in the process that the opaque side of the quartz cell is facing the left side of the MicroSampler as you re facing the unit Disposable filter Notes amp Tips 1 Some DynaPro users prefer to centrifuge rather than filter their samples If you choose this approach 10 15 minutes at 3000 RPM is usually sufficient to move dust particles to the bottom of the centrifuge tube PROTEINSOLUTIONS www protein solutions com 46 Checking the hardware configuration Prior to starting an experiment in DYNAMICS V6 you should always check to insure that the default hardware configuration is consistent with the experiment you are about to conduct To check the hardware configuration select the Hardware Node in the Tree View The current configuration will be displayed in the properties table to the right of the Tree View New experiments are opened with the File New command on the main menu bar Current list of user defined analog detectors Property Value empty Host 99 D 50 Host Serial Number 99 D 50 Generic UV 281 Optics LSR 045 Optics Serial Number LSR 045 Waters RI Parameters Aneioc rout 1 BNC pH probe Fixed u SSO NP Y Instrument Analog Input 2 empty Solvent Analog Input 3 empty Supported RS232 Sample aa UserDefined Bu Serial COM
97. rburst Polymers Tol Int Cnt s The scattering intensity in counts second of a toluene standard The value is used to calculate the Rayleigh ratio of the analyte in static light scattering applications Tot Inj Vol mL The total volume of titrant injected up to the designated measurement in units of milliliters This property is only available if a titrator is present in the hardware configuration The value is determined by the titrator algorithms and is not user editable Vol mL The milliliter sample volume If a titrator is present in the hardware configuration only the Vol mL value for measurement 1 is user definable all other volume values are dependent upon the titrant injection volume i e the Inj Vol mL and are defined by the automatic titrator routines Note that if the user changes the volume value for measurement 1 during or after the titration the values for subsequent measurements will be updated accordingly Measurements Node Table Settings Pd The polydispersity of the sample determined using a Cumulants analysis Abs Al The sample absorbance determined from the Abs Al vs UV AI calibration curve for an analog UV detector connected to the BNC jack Amp The amplitude intercept of the non normalized intensity autocorrelation curve Value varies from 0 to 1 Baseline The measured value of the normalized intensity autocorrelation curve at the last channel used Values of 1 000 indicate that t
98. rmation regarding your particular DynaPro instrument If there are no hardware components stored in the ini file the software will request the information from the user by presenting the Add Hardware window The hardware configuration settings for your DynaPro are printed on the inside cover of your DYNAMICS V6 Installation Disk After entering the settings click the OK button and the information will be saved to the Dynamics ini file for future reference PROTEINSOLUTIONS www protein solutions com 27 28 Original Hardware X DYNAMICS V6 cannot detect any hardware Please describe you system You find the hardware settings for your system on the inside cover of the DYNAMICS V6 installation disk Host Serial Host Model H Optics Serial Optics Model H Wavelength nm ot Min Lys Cone mg ml fo Toluene Intensity Cnit s BR Water intensity Cnt s fo P IFlex Laser I Temperature Control I Flow Kit J Stir Kit I UltoSpec 4000 I Titrator Connecting to the DynaPro Prior to collecting light scattering measurements DYNAMICS V6 must be connected to the DynaPro host unit The connection is made in the Hardware Node of the Experiment Window To open an Experiment Window click the new file icon on the main tool bar or select the File New option on the main menu bar Open New Experiment Ose a amp To configure the hardware select the Hardware Node in the Tree View on the left hand s
99. rrelation View The Regularization View PROTEINSOLUTIONS www protein solutions com Formatting tables Saving Workspace settings Defining solvents in DYNAMICS V6 Defining user defined parameters Application Options Using DYNAMICS V6 Troubleshooting PROTEINSOLUTIONS www protein solutions com 92 93 94 94 95 96 DYNAPRO TECHNICAL MANUAL DynaPro and Dynamics are trademarks of Protein Solutions Inc a Ey I509001 RERSTEREI ComPaNyY Protein Solutions Ltd is an ISO9001 registered company certificate number 8381 PROTEINSOLUTIONS www protein solutions com Statement Of Warranty Protein Solutions warrants that the fabrication and components of the DynaPro Molecular Sizing Instruments are free of manufacturing defects for a period of 12 months from the date of installation At its discretion and as deemed necessary Protein Solutions will provide a replacement part repair the product at a Protein Solutions facility or repair replace any defective item including parts labor and shipping All costs for such replacement or repair will be covered by Protein Solutions during the 12 month warranty period This warranty will extend only to repairs required in the course of normal operations Protein Solutions will not be obligated to repair any equipment which in its sole judgment has been altered or damaged by unauthorized persons i e not in the employ of Protein Solutions or by carrying out work w
100. see the correlation curves select an Acquisition or Measurement in the Tree View and then click the Correlation View button on the Experiment Window tool bar Display Correlation View dynamics MSX qa3 exp i File Edit View Window Help DER ytelse Bibs E a a 21 E MSX qa3 exp E Hardware Correlation View display options color coded for olx Average correlation curve for Meas 1 lelx excluding Marked acquisitions r Control Panel distinguishing data in the correlation curve figure Current Channel Cut Offs are set to 1 and 120 on in Parameters Click axis to M Correlation Curve s E Measurements 14 change between IV Measured Baseline Meas 1 a Li a in amp Log scales I Theoretical Basel i i Acq1 S 13 8 en Use these options to display Acq2 EDI I Channel Cut Offs 1120 4 the best fit from a Cumulants Acq 3 en SER p Average Acad z and or Regularization ana ysis Acq5 5 IV Cumulant fit c Aab 10 I Regularization fit Use this option to either cq A Acad 0 10 100 00 1 00E 05 1 00E 08 T Merked Curve s hide or display Marked Acq9 Time us lie correlation curves Acq 10 89 Red color indicates Acq 3 is currently Marked as an outlier 0 020 0 010 a 0 000 0 010 0 00 1 00E 05 1 00E 08 Time us Correlation curves selected with a left click are noted with a green color Residual Graph showing the difference bet
101. solutions com 58 Using DYNAMICS V6 Overview This tutorial is the fifth part in a series of tutorials designed to introduce the new user to the DynaPro Molecular Sizing Instrument and the DYNAMICS V6 software suite The objective of the tutorial is to familiarize the user with the basic structure and functions of the software For more details and help with special applications you should also see the Application Oriented Software tutorial What is DYNAMICS V6 In earlier versions of DYNAMICS the objective of the software was to simplify the collection analysis and interpretation of the time dependent and time average intensity of light scattered from a solution of particles i e to minimize the difficulty typically associated with static and dynamic light scattering experiments With DYNAMICS V6 the scientists and engineers at Protein Solutions have reached far beyond light scattering and set their sights on developing a more complete laboratory PC assistant a software package that could not only increase the speed at which light scattering data was collected but also increase the amount of information given to the user from the same experiments The goals in the DYNAMICS V6 development project were to develop a package that could 1 Collect analyze and store data received from other common laboratory instruments 2 Enhance the macromolecular characterization capabilities of the DynaPro Molecular Sizing Instrument by integrati
102. specific to the selected node in the Tree View will also be available in the display side PROTEINSOLUTIONS www protein solutions com 62 Data grouping in the Tree View The Tree View in DYNAMICS V6 is used to select groups or categories of information for viewing in the display side of the Experiment Window There are four main nodes in the Tree View Hardware Parameters Event Schedule and Measurements Information Data associated with selected node in Tree View Property Value Host 99 D 50 Host Serial Number 99 D 50 X Optics LSR 045 Optics Serial Number LSR 045 UltroSpec 4000 OO pr empt Ei Parameters 09 np x ia User Defined Analog Input 2 empty ed Solvent Analog Input 3 empty r Calculations Serial COM 1 UtroSpec 4000 Event Schedule Bl Serial COM 2 empt eI Measurements m x Connection Status Not Connected onnec Disconnect Tree View Node specific command functions Hardware Node This node contains all the parameters and settings necessary for describing the instrumental hardware associated with the experiment Hardware components such as external detectors titration units etc are added and removed at the Hardware Node using the list boxes in the properties table Parameters and settings for a specific piece of hardware are viewed by selecting the hardware component in the Hardware N
103. stablish communications between the computer and the temperature control module in the DynaPro optics block use the RS232 cable included with the instrument to connect the two in the fashion shown in the figure below applies only to MicroSamplers with temperature control upgrades Note that COM port selection on the back of the computer is irrelevant When the optics block is added to the Hardware Configuration in DYNAMICS V6 the software will search all available COM ports and assign the appropriate COM port settings The same is true for other supported RS232 devices 34 58 ge Bo gt ze E Ere co DO NOT Connecting analog digital I O devices The DynaPro and DYNAMICS V6 support the integration of external analog digital I O devices such as HPLC detectors injectors and pH meters into the experimental setup and procedures Communications between the software and these types of external devices are handled via the BNC jacks on the back of the host unit Note that A D connectors are only available on host units containing a Flow Kit upgrade PROTEINSOLUTIONS www protein solutions com 24 Notes amp Tips 1 DYNAMICS V6 cannot determine which analog jack is being used for which external module When connecting external devices you should note the associated with the BNC jack so that the correct entry can be made in the Hardware Node in the software 2 The 24 bit A D chip on the DynaPro control board is des
104. t the fit of the inserts is rather tight After opening the boxes you should find various cables a Quick Start Manual a software CD and a quality assurance Microsphere standard Remove all loose items and lift the instruments Host unit and MicroSampler out of the boxes It might be helpful to place the box on its side and rock the foam inserts out Inspect the instruments for visual shipping damage and report obvious trouble to our technical support department Then place the Host unit and the MicroSampler close to the computer which will be used to operate the DynaPro While we at Protein Solutions strive to make the DynaPro as sturdy as possible it is a precision instrument and needs to be carefully handled to insure the laser alignment remains optimized We have specially designed the DynaPro packing material to minimize damage arising from unavoidable rough handling during shipping Please do not discard this packaging material In the event that you need to return the instrument to Protein Solutions for either a repair or an upgrade we ask that the unit be shipped in the original packaging material PROTEINSOLUTIONS www protein solutions com 17 Stacking the DynaPro modules Typical DynaPro instruments consist of two instrument modules the Host unit and the MicroSampler The Host unit is the controller and data collection component for the suite of hardware modules and accessories that can be integrated into the DynaPro M
105. t a single correlation curve Larger acquisition times result in better signal averaging but also increase the likelihood of a dust event occurring during the course of the acquisition Flow Rate mL m The flow rate of the HPLC pump in units of mL min This parameter is only available if an HPLC pump is present in the hardware configuration and is used along with the analog detector Offset Vol mL to calculate the time difference in the HPLC peak shift routines Laser Power The percentage of the available laser power that is to be used or was used during the measurement Read Interval s The amount of signal averaging time in seconds for instantaneous readings of the scattering intensity and analog detector signals Set Temp C The user defined target temperature for temperature controlled optics blocks Solvent Parameters Node Abs Al Baseline The solvent baseline absorbance for an analog UV detector connected at the BNC jack on the back of the host unit This parameter is only available if an analog PROTEINSOLUTIONS www protein solutions com 73 UV detector is present in the hardware configuration and the value is used as a background subtraction for concentration calculations via Beer s law in HPLC applications Cauchy Coef nm The slope of a plot of vs A in nm for the solvent The parameter is used along with the Rfr Idx 589nm amp 20C to calculate the refractive index of
106. t air temperature of the day Remote Dry Air Purging A self contained dehumidifying circuit is fitted as standard for temperature controlled MicroSamplers Humidity within the sample cell enclosure is continuously monitored and controlled when the instrument is operational The system circulates air through a closed PROTEINSOLUTIONS www protein solutions com 10 loop network of tubes and desiccant drying material An air pump is activated every time the instrument is switched on if the lid accessing the sample cell is lifted or if the relative humidity within the enclosure exceeds 85 When the pump is activated air passes through a desiccant drying column where the blue drying granules change color to pink as they absorb moisture Some users may wish to use an external source of dry gas dry Nitrogen for example instead of replacing the desiccant granules If requested with the purchase the MicroSampler can be fitted with two optional 4mm push in pipe fittings at the rear of the instrument for remote air purging The red plugs are removed by pressing the black retaining ring while pulling the plug straight out A dry gas line can then be connected The system is an open circuit design containing a one way valve where gas will flow only from the IN side to the OUT side The dry gas line may be left under pressure safely and is controlled by an internal solenoid valve This solenoid allows gas to pass through the networ
107. t this level is lost Tree View x Read 5 10492 when the file is closed Read 6 20526 Read 10502 Read 8 10495 Read 9 10453 Read 10 10480 www protein solutions com 88 Consider for the example the marking scheme shown in the above below At the main Measurements Node the average 20849 Intensity for all the Acquisitions in Meas 1 has been marked as an outlier At the Meas 1 node the average 19633 Intensity for all the Readings in Acq 4 and the acquisition data for Acq 2 have been marked At the Acq 1 node for Meas 1 the Intensity for Read 6 20526 is also marked When the experiment file is saved the marking associated with the Intensity for Meas 2 the Intensity for Meas 1 Acq 4 and the acquisition data for Meas 1 Acq 2 will be saved The marking associated with Meas Acq 1 Read 6 however will be lost The Correlation View The intensity correlation curve is the raw dynamic light scattering data from which the hydrodynamics properties calculated within DYNAMICS V6 are derived For monomodal single size samples the correlation curve should be a smooth exponential with an amplitude intercept ranging between 1 1 and 2 0 and baseline of 1 00 While the SOS sum of squares error for a Cumulants fit amplitude and baseline are fairly good parameters for judging the goodness of the correlation curve it s typically a good idea to also look at correlation curves to insure that all are reasonable To
108. tensity Model Spheres View Curves af JE legend T Suppress First At low temperatures the Depending upon the 44 SIMPLE BATCH EXPERIMENTS PROTEINSOLUTIONS www protein solutions com Simple Batch Experiments Overview In The Microsphere Standard tutorial the batch measurement was conducted using a predefined set of experimental parameters It s unlikely however that the experimental parameters appropriate for the Microsphere standard will also be appropriate for all batch experiments In the Simple Batch Experiments tutorial the user is introduced to the parameter entry procedures used in DYNAMICS V6 The scope of the descriptions given in this tutorial are still introductory For more detailed descriptions of the options available in the software see the Using DYNAMICS V6 tutorial Preparing the sample When preparing a sample for light scattering measurements it is always a good idea to filter the sample as it is introduced into the cell First insure that the cell is thoroughly clean and dry Then insert an appropriate filter in line between the sample syringe and the syringe needle insert the needle all the way to the bottom of the quartz DynaPro cell and dispense only enough sample to just fill the cell window You should then cap the cell and wipe any fingerprints or smudges from the surface using a piece of lens paper laboratory tissues are not recommended due to the possibility of sc
109. ter are applied to the data set Notes amp Tips 1 You can still Mark data manually while the Experiment Window is in the recording mode even if the Real time data filter option is set to True However if you wish to preserve the manual marking you should insure that the Keep manual marking option is enabled in the Data Filter window A note on saving Marked data One of the advanced features of DYNAMICS V6 is the ability to group an unlimited number of measurements sets of acquisitions into a single experiment file As a consequence of this versatility however the saving of all data marking permutations is problematic While you can apply manual or automatic data marking at any level of the Tree View while the experiment file is open when the file is saved only the marking at the main Measurements Node and the Measurement Meas level is saved Main Measurements Node Item Intensity R Meas 1 10562 3 81 Marking at these levels is Meat 20249 3 97 saved when file is saved Meas 3 10757 3 75 Meas 1 Node E Expl Item Intensity R Hardware Acg 1 10481 389 F Evert Schedide Acq2 10652 685 E Measurements Acq 3 10554 3 78 E Meas 1 Acq4 19633 382 Acq Acq 5 10547 380 Acg2 Acq3 Acq 1 Node Boa 4 Item Intensity R Acq5 ee E E Meas 2 Read 1 10495 G Meas 3 Read 2 10558 Read 3 10451 3 Read4 10489 _ Marking a
110. the drivers If you encounter this problem please contact technical support at Protein Solutions for assistance Starting DYNAMICS V6 DYNAMICS V6 is the instrument control and data analysis software for the DynaPro Molecular Sizing Instrument The software can be launched by double clicking the icon on PROTEINSOLUTIONS www protein solutions com 26 the Windows desktop or by single clicking the icon on the Windows Task Bar Start Programs Dynamics Dynamics V6 DYNAMICS Expl File Edit View Window Help Cee Ae For Help press F1 Notes amp Tips 1 DYNAMICS V6 saves experiment files with a exp extension A short cut for opening saved experiment files is to left click the file and then drag it to the top of the DYNAMICS V6 icon on the Windows desktop Once the file is released the software will open with the selected experiment file loaded 2 Saved experiment files can also be opened directly into DYNAMICS V6 by simply double clicking the exp file If you find that Windows does not recognize the exp extension you can update Windows by selecting the Dynamics V6 option in the File Types list box that Windows presents when a file of unrecognizable format is double clicked 3 In contrast to previous versions of Dynamics DYNAMICS V6 does not automatically connect to the DynaPro when the software is loaded Original Hardware Configuration When DYNAMICS V6 opens it looks to the control file Dynamics ini for info
111. ty Control testing and in house instrument performance level diagnosis In order to accommodate field testing and the self diagnosis routines incorporated into DYNAMICS V6 Protein Solutions incorporated a 5 mL sample of the Microsphere standard into the standard shipping list for each DynaPro instrument beginning in November 2000 In this tutorial the procedures for preparing and measuring the Microsphere standard are presented Instructions for comparing your results for the standard to those collected at the Protein Solutions Quality Control Laboratory are included Should you encounter problems with either measuring the standard or interpreting the results please contact the Protein Solutions Technical Support team at 1 888 242 2848 or visit the Technical Support section of our website at www protein solutions com Notes amp Tips 1 The Microsphere standard was designed for ambient temperature stability and should be stored at room temperature Refrigeration will lead to irreversible aggregation and effectively render the standard useless as a diagnostic tool 2 Microsphere Quality Control limits for the various DynaPro units MS X MS 800 and LSR are given in the Interpreting Microsphere results section of this tutorial Preparing the Microsphere standard The formulation of the Microsphere DLS standard was designed to allow the standard to be filtered through a 0 02 um Anotop filter The dropper bottles in which the Microsph
112. type in the Correlation View is linear for the Y axis and logarithmic for the X axis If you re more familiar with a different style correlation curve presentation switching between linear and logarithmic scales can be accomplished by clicking the appropriate axis 2 The channel cutoffs for the intensity autocorrelation curve can be changed from within the Correlation View To change the cutoffs check the Channel Cutoffs option box to display the cutoffs center the mouse cursor over the cutoff to be changed mouse cursor will change to a double headed arrow and then click and drag the cutoff to the new value As the cutoff is moved the numbers in the Channel Cutoffs option box label will change accordingly 3 The Export option in the Correlation View right click menu can be used to export the data for the correlation curve and residual figures in text format to a user defined file The Regularization View In DYNAMICS V6 the Regularization View shows the calculated size distribution for the correlation curve associated with the measurement or acquisition selected in the Tree View The distribution can be presented in terms of either Ylntensity or Mass on the Y axis and PROTEINSOLUTIONS www protein solutions com decay time diffusion coefficient radius or diameter on the X axis The Model list box can be used to apply the appropriate form factor in the Intensity to Mass transform for non spherical particles Intensity is i
113. u re working with larger particles such as gold and silver colloids macromolecular assemblies and some polymers the scattering from the sample itself is sufficient to counter any scattering contribution from dust particles assuming reasonable care is taken during your sample preparation i e filtered solvent clean cell etc As such number fluctuations arising from dust are less likely to be observed since the scattering contribution is virtually negligible 4 The upper size range limits for the DynaPro Molecular Sizing Instrument is dependent upon the model type 50 nm radius for the high sensitivity MS 800 1 um radius for the high sensitivity large size range MS X and 1 um radius for the high concentration large size range LSR Using the MicroFilter Kit The MicroFilter Kit is a DynaPro accessory developed at Protein Solutions to facilitate filtration of low volume samples The MicroFilter Kit comes complete with all the tools necessary for performing low volume filtrations including a 100 uL syringe a metal housing for the filter disk replacement seals tweezers for handling the filters and an ample supply of Whatman Anotop filters The recommended procedures for using the MicroFilter Kit are as follows 1 Unscrew the metal housing separating it into two parts 2 Remove the two seals rubber O rings PROTEINSOLUTIONS www protein solutions com 33 34 3 Thoroughly rinse all parts with DI water and then dr
114. uchy Coefficient defines the dependence of the solvent refractive index on the wavelength and is used in conjunction with the Ref Ind 589nm amp 20C property to calculate the solvent fi at the DynaPro laser wavelength Temperature Model The Temperature Model is used to describe the temperature dependence of the solvent viscosity and refractive index Intensity The solvent intensity is used in Correlation Noise Factor CNF calculations and in the production of static MW Zimm plots Note that the Solvent Intensity is not a required parameter for dynamic light scattering measurements Notes amp Tips 1 User Defined solvents can be named and saved to the DYNAMICS V6 Solvent Database in the Tools Parameters Solvents window 2 The influence of the Cauchy Coefficient on dynamic light scattering results is small If you are unsure use the value for water 3119 User defined experimental parameters Experimental descriptors are parameters typically user defined that are used to describe the experiment e g sample lot pH and user s name In DYNAMICS V6 these descriptors are stored in the User Defined Node in the Tree View Exp3 Exp3 EH Hardware Parameters Fixed Instrument Solvent Sample 3 Enter the value of Measurements the parameter here Measurement 1 Select the parameter to be added here I Parameter Incubation t
115. ucing the laser power if the scattering intensity is gt 8 million Cnt s For the high sensitivity DynaPro MS 800 units you may trigger the Detector Protector when measuring the Microsphere standard If you do the laser power can be reduced using the following procedures 1 Stop the experiment or recording 2 Open the Instrument Control Panel by clicking the host unit icon on the Experiment Window tool bar 3 Reduce the laser power to 50 with the slider bar 4 Reconnect the instrument at the Hardware Node in the Tree View 5 Restart the data recording by clicking the green Record Button Interpreting the Microsphere results The results for your Microsphere test are best interpreted by direct comparison to the Microsphere quality control results for your instrument The open the quality control experiment select the File Open option on the menu bar and find the serial qa3 exp file in the Protein Solutions Quality Control folder For typical software installations the path to the file will be c Program Files Protein Solutions Quality Control serial qa3 exp After opening the quality control file you can display the results in graphical format by selecting the Trace View button on the tool bars of both experiments PROTEINSOLUTIONS www protein solutions com 42 DYNAMICS V6 F dol x File View Tools Wi x Deu kr Display Trace View E
116. ure in the software can be used to automate temperature control for sampling sequences The maximum period of time to achieve a stable temperature after ramping between the upper limit of 60 C and the lower limit of 4 C should be no more than 7 minutes This period of time assumes a humidity of up to 75 and an external ambient temperature of no more than 25 C If you observe longer stabilization time periods please contact technical support at Protein Solutions In practice it takes only a few minutes to reach and stabilize target temperatures Air circulation is maintained by the provision of circular ducts on all sides of the MSTC enclosure Do not allow these ducts to become obstructed in any way or the performance of the instrument may be impaired Fluid Cooling Heating Enhancements The temperature controlled MicroSampler can be constructed with fluid heating and cooling features provided these have been requested when the instrument is purchased The provision of 2 connectors at the rear of the instrument enclosure allows for fluids to be passed through the heat sink matrix of the sampling chamber thereby facilitating the use of an external temperature bath and an increased operational temperature range for the DynaPro The system is pressure tested to local mains water pressure and is specified to a maximum flow rate of 100 mL s When operated at this flow rate the cooling performance is improved to 45 C below the ambien
117. used for communications between DYNAMICS V6 and the DynaPro optics block through the host unit Notes amp Tips 1 The 24 bit A D chip on the DynaPro control board is designed to handle 0 to 2 5 V analog signals Negative signals can damage the chip Prior to connecting an external detector please insure that the specified output signal for the detector is within the acceptable specifications 0 2 5 Volts for the DynaPro If you are unsure please contact Protein Solutions for advice Connecting the optics block WARNING Room light can destroy the avalanche photodiode detector You should always insure that the power to the host unit is turned off prior to connecting and or disconnecting the fiber optics The DynaPro operates by illuminating the sample with coherent high intensity laser light Light scattered from the sample is monitored at a 90 degree angle to the incident beam Fiber optic cables are used to guide the incident light and the scattered light to and from the sample cell both the laser and the APD detector are contained within the DynaPro host unit Hence there are two fiber optic connections that need to be considered when setting up the DynaPro instrument the fiber optic from the laser to the cell and the fiber optic from the cell to the detector The laser fiber is color coded either red or green and needs to be connected to the port labeled Laser Aperture on the host unit The detector fiber
118. w In other words DYNAMICS V6 allows the user to view and even perform functions on data from another measurement while saving new data A new measurement category is automatically created in the Measurements Node every time the Experiment Window is placed in the recording mode New acquisitions cannot be added to an existing measurement The acquisition time and laser power are fixed parameters for a given measurement When the Experiment Window is in the recording mode the Acquisition Time and Laser Power functions in the Instrument Control Panel are disabled To adjust these parameters you ll need to stop recording make the adjustments and then re start the recording process Displaying data in the Grid View The Grid View in DYNAMICS V6 is used to display data from the Measurement Node in a table format For users familiar with earlier versions of Dynamics the Grid View incorporates all the elements of the Cumulants datalog with a variety of new features such as unit conversions user defined table formatting right click menus and copy paste functions PROTEINSOLUTIONS www protein solutions com 82 Click here to show the acquisition data and the average of the instantaneous readings w L u 7 collected during each acquisition El MSX ga3 exp H Hardware D Time Intensity Amplitude D R Baseline SOS E Parameters 6 Cnt s
119. ween the measured and fitted data For Help press F1 F NN 2 A variety of overlay and display options are available for selection in the Control Panel for the Correlation View Overlays are color coded for ease of viewing and are activated by checking the appropriate box in the Control Panel Color coding is also incorporated into the PROTEINSOLUTIONS www protein solutions com Residual display below the correlation figure The Control Panel and Residual can be hidden by deselecting the option in the Correlation View right click menu Note that the figure can also be copied to the Windows clipboard using the Copy command in the right click menu i File Edt View Window Help Note the absence of the Control Panel and la x Os amp amp 2 Residual hidden by un checking the options bl PK la Bl in the Correlation View right click menu El MSX qa3 exp 6H Hardware E Parameters B Measurements El Meas 1 cq1 Acg2 Copy Ctrl C Export Data Filter Figure settings Control Panel Residuals Acg 3 Acq 4 Acg5 Acg6 Acq Acq 8 Dynagtept sity Autocorrelation Bad correlation curve likely a consequence of dust during the acquisition period The red color indicates Acq 3 has ER been marked as an outlier 1 00 1 00E 03 1 00E 05 1 00E 07 Time us For Help press F1 Notes amp Tips 1 The default axis
120. whether or not an automatic titrator is available Min Lys Conc mg mL The original equipment minimum lysozyme concentration in units of mg mL measured in the Protein Solutions Quality Control Laboratory This parameter is used to calculate the molecular weight dependent minimum sample concentration for the original equipment in the Optimization Calculator Tools Calculators Optimization PROTEINSOLUTIONS www protein solutions com 66 Toluene Int Cnt s The scattering intensity in counts per second for a toluene sample measured in the Protein Solutions Quality Control Laboratory This parameter is used for original equipment calculations in the Optimization Calculator and is also the default value for the toluene intensity Tol Int property in Sample Node used in static molecular weight calculations Water Int Cnt s The scattering intensity in counts per second for pure water DynaPro host units and optics blocks are edited defined and or deleted using the Host Units and Optics Blocks radio buttons in the Tools Parameters Hardware window Values are edited by double clicking the appropriate cell or selecting a new entry in the drop down list box New units are defined by selecting the New option in the Host Optics Unit list box Edit Hardware z p x Host Unit properties Property Value Original Equipment Host Unit test Ho iS Serial Number test C Optics Blocks Mo
121. xp1 ojx Mm Cu e Gay TT IEE El ee Intensity and radius results Expl n r Control Panel a El Hardware for your Microsphere test y asis Parameters 2800 Tine Measurements 55 EB Meas 1 my Y Axis 2500 G 50 a xy E o 2 5 2400 s Er 3 S E 2200 r 10 20 30 4 50 60 Time s gt ar lwj ej m 99 180 ga3 exp Zontrol Panel Microsphere Quality Control results for your DynaPro Hardware x Anis E Parameters Time Measurements 55 z a YAris 2600 Intensity MT amanar sh ra nd Left Right Scalar wu 4 2400 Intensity kCntis 2200 o o o 10 20 30 4 50 6 Time s For Help press F1 The radius result displayed in the Grid and Trace Views is the radius calculated from a Cumulants analysis which assumes a monomodal single particle distribution The DynaPro however is sensitive enough to detect the presence of the surfactant stabilizer added to the formulation The weighted average calculated by the Cumulants algorithm then will be slightly lower than the 6 nm reported size of the Microsphere Typical Cumulants radius values for the Microsphere standard are 4 3 0 3 nm The magnitude of the intensity will vary with the DynaPro model and the laser power If you conducted your experiment at the same laser power and same temperature as the quality control test the measure
122. y 4 Seat the two seals back into the two halves of the metal housing 5 Using the tweezers place a filter disk into the needle half of the metal housing See the Notes below for tips on getting the filter into the correct position 6 Load the syringe with a small amount of solvent and then insert the syringe needle into the Teflon needle guide in the filter housing Rinse the filter with solvent from the syringe 7 Remove the syringe from the housing and dispense any solvent remaining into a waste container 8 Reload the syringe with a small amount of sample and then reinsert the syringe needle into the Teflon needle guide in the filter housing 9 Depress the syringe plunger enough to dispense 1 2 drops Two drops are sufficient to displace any solvent that was wetting the filter disk 10 Load the quartz cell and collect your light scattering data 11 Remove the syringe from the metal filter housing and dispense any remaining sample back into your sample container 12 Disassemble the metal housing and thoroughly rinse and dry all components before placing them back in the MicroFilter case Notes amp Tips 1 After placing the filter into the filter housing the seating of the filter is difficult to adjust using only the forceps Twisting the Teflon housing will help seat the filter in the proper location within the rubber seal PROTEINSOLUTIONS www protein solutions com Spinning the sample Rather tha

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