MicroCal VP-ITC User Manual Download Page 5

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Section 1: VP-ITC – General Information

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iii

Summary of Contents for VP-ITC

Page 1: ...35 3 1 Designing ITC Experiments 36 3 2 Using the ThermoVac to Degas Samples 40 3 3 Cell Loading 41 3 4 Loading the Auto Pipette 42 3 5 Choosing The Run Parameters 45 3 6 Carrying Out Experimental Run...

Page 2: ...VP ITC MicroCalorimeter User s Manual This page was intentionally left blank...

Page 3: ...iptions toward a better understanding of the intricacies of the instrument Section 5 contains important information for maintaining the instrument performance Section 6 provides tips for optimizing th...

Page 4: ...ems typically correctable by the operator For performance issues such as these it is recommended that customers carry out the following minimum diagnostic steps prior to contacting MicroCal for servic...

Page 5: ...d on the VP ITC instrument Caution Read the instruction manual before operating Instrument supply ON Instrument supply OFF Protective Earth Ground Terminal Origin is a registered trademark of Origin L...

Page 6: ...or should always follow proper laboratory procedures in handling e g wear safety glasses and protective clothing and disposing of such materials and allow the internal cells to cool down to room tempe...

Page 7: ...MS windows averaged over 1 hour 1 nanocal sec 4 nanowatts Baseline Constancy average one hour time intervals 310 rpm stirring 5 nanocal sec 20 nanowatts Minimum Response Time 15 seconds Operating Temp...

Page 8: ...y Output Secondary Data Connection only Protective Earth Terminals Internal external marked Mode of Operation Continuous Classification Class I Atmospheric Specifications Operating Temperature 10 to 4...

Page 9: ...r controller and VP ITC instrument and disconnect the mains power supply cord before cleaning the case To clean the instrument case use a damp lint free cloth Avoid abrasives or solvents that can perm...

Page 10: ...C Electromagnetic Compatibility and Low Voltage Directive 72 23 EEC Electrical Safety The following tests were carried out Directive Generic Standard Referenced standard 89 336 EEC EMC EN 55011 Group...

Page 11: ...ting up the VP ITC instrument and accessories It ends by providing information about the ThermoVac instrument This section is arranged as follows Section 1 VP ITC System Introduction 1 1 1 VP ITC The...

Page 12: ...e sample cell This signal is sometimes referred to as the feedback power used to maintain temperature equilibrium Calibration of this signal is obtained electrically by administering a known quantity...

Page 13: ...anges are 2 5 and 5 volts ITC instrument control is accomplished through the second interface board a 16 bit D A digital to analog converter board Programmed by the VPViewer software this board will r...

Page 14: ...dips and spikes Although the power filtering in the VP ITC instrument is adequate for most laboratory environments some disturbances may affect the performance of the instrument and it may be necessar...

Page 15: ...C Cell ASS130140 1 Pipette Stand ASS130150 1 Injector Stirrer Base mounted on top of ITC cell unit INT100010 1 Computer Controller call for part number 1 Printer optional SOF100040 1 VPViewer software...

Page 16: ...30230 1 Tube holder white for pipette stand MSP13057 1 Plunger Tip Pusher tool MSP13056 1 Plunger Tip Puller tool MSP13058 1 Guide sleeve MSP13043 1 Threaded Cap FUS100010 2 Fuses 1A 250V 5x20mm Slo B...

Page 17: ...ly If a contortion of the cable appears necessary to accommodate the appropriate connections contact MicroCal for assistance Connect each of the two Flat Main Cable connectors labeled J1 J2 to their a...

Page 18: ...plug Please Note It is strongly recommended that the VP ITC be provided a dedicated AC power line with no other equipment being powered on that line see page 4 for more information about selecting a l...

Page 19: ...plunger is in the open port position all the way up Failure to follow this recommendation may result in a bend in the pipette screw Even a slight bend will result in improper operation of the pipette...

Page 20: ...the pipette holder attached to the side of the VP ITC Bring the pipette injector directly above the syringe holder start bringing the injector down while observing the plunger tip of the pipette inje...

Page 21: ...and cleaning the cells you have been provided with the ThermoVac accessory a device for thermostatting and degassing samples The unit is capable of thermostatting a sample at any temperature from 0 t...

Page 22: ...n for approximately 5 minutes recommended time for degassing samples then the pump will shut off If you desire to remove the vacuum before the preset time you must turn off the main power switch locat...

Page 23: ...ed and on the temperature of the sample the vacuum strength might be enough to boil the sample and subsequently change the concentration and or spill the sample This may be avoided by adjusting the st...

Page 24: ...hat reason skip this section and proceed to the Section 4 tutorials for a hands on approach to learning the software though simple experiments While performing the tutorials the user should refer to t...

Page 25: ...Origin for data analysis simultaneously with VPViewer as well as most other Windows applications In general VPViewer is a multi tasking application with some subtle limitations If VPViewer detects a c...

Page 26: ...l power and the Power LED to come on Likewise after closing VPViewer it will require a minute or so for the cell power and Power LED to turn off When the VPViewer application is first opened it will g...

Page 27: ...aunch VPViewer the Main Window of VPViewer is displayed as shown below VPViewer Data Channel Display By default VPViewer displays three data channels and the status of the instrument Instrument Status...

Page 28: ...d the DP value should be positive and stable The power level of the equilibrated DP signal should be close 1 uCal sec to the Reference Power setting as set prior to starting the run DT deg C This text...

Page 29: ...ing on page 72 for details on y axis calibration Help Help Allows access to the help menus At the time of release of this User s Manual the on line help was still under development About Provides spec...

Page 30: ...is available only when the VP ITC is in a non idle state Note Only run parameters that have not yet been applied on the run in progress may be updated all others will be ignored e g after the stirrin...

Page 31: ...ere is a check mark next to the No Check Temp option in the ITC Equilibration Options group see below the entered temperature will be ignored and the experimental temperature will take place at the cu...

Page 32: ...sh a baseline prior to the first injection Syringe Concentration mM Enter the mM concentration of the solution in the syringe This value is stored in the data file header and used in Origin for ITC da...

Page 33: ...st possible time put a check mark in each box No Check Temp When this check box is selected the Cell Temperature C text box entry will be ignored and the experiment will start at the current cell temp...

Page 34: ...turn to the baseline after an injection peak deflection Typical values for this parameter range from 180 300 seconds depending on the size of the peak and the kinetics of the reaction Filter Period se...

Page 35: ...Port This button is for positioning the plunger below the fill port after filling the pipette with your sample When you click on the button the injector screw will extend so the plunger tip is just be...

Page 36: ...ing cell cleaning can often times improve the effects of the cleaning Pulse Control Allows for manually administering a DP calibration pulse While this is not the most thorough method of checking the...

Page 37: ...nge the current data file path double click on the existing path text and a dialog box will open allowing you to change the path The desired data directory must already exist in order to select it fro...

Page 38: ...d placing data files in different folders for multiple users See section 7 2 Customizing VPViewer starting on page 82 for more information Y Axis Scale The Y axis group allows for changing of the curr...

Page 39: ...tools may be used to remove the old plunger tip Install New Tip Click this button to position the injector plunger in a retracted position so that the Tip Pusher Tool may be used to insert a new plung...

Page 40: ...ides viewing access to the calibration constants for your particular VP ITC instrument Shown below are calibration constants that are typical of the VP ITC This window is password protected and should...

Page 41: ...to perform data analysis This section is intended to detail those features that are contained in the VPITCPLOT OPJ data display project of Origin Please refer to the Origin User Manual for a complete...

Page 42: ...determined by the entry in the Full Scale Auto View 2 text box see Edit ranges below Saved View 1 When you click on this button the y axis will be scaled from the value entered in the Saved View 1 Y M...

Page 43: ...accomplished when the cell temperature is within 0 1 degrees of the entered temperature and the DeltaT temperature difference between cells and adiabatic jacket is less than 0 001 Deg To save equilib...

Page 44: ...g the syringe The start of the stirring will cause the DP signal will deflect downward then the signal will increase but stabilize at a level slightly lower than the non stirring baseline The instrume...

Page 45: ...h simple experiments While performing the tutorials the user may refer to this section if further detail is desired about a specific operation After performing the tutorials it is advised to read thro...

Page 46: ...binding By non linear least squares fit of calorimetric titration data the parameters K Ho and n are determined directly in a single experiment and Go and So may then be calculated Titration calorimet...

Page 47: ...duced by decreasing Mtot i e holding K Ho and other parameters constant the drop near the equivalence point becomes broadened and the intercept at the Y axis becomes lower than the true Ho In the limi...

Page 48: ...erimental K window so the concentration range available in the K window becomes 3 x 10 6 M 10 3 Although any value within this range is acceptable it would lead to better estimates of parameters to ch...

Page 49: ...ay change due to titration of ionizable groups on the ligand If this happens then the ligand solution should be back titrated carefully until the pH is identical to that of the macromolecule solution...

Page 50: ...use Please note When running experiments below ambient temperature the temperature of the sample should be such that after loading into the cell it is at or below the experimental temperature This wi...

Page 51: ...adjust the bleeder valve Turning the adjusting knob clock wise will increase the vacuum while a counterclockwise turn will decrease the vacuum strength Place the Vacuum cap on top of the sealing o ri...

Page 52: ...top of the metal cell stem and remove the excess solution Fill the reference c Sample reservoir excess water To prevent sample overflow into the reference cap the reference cell with the Reference Plu...

Page 53: ...syringe to draw up the titrant solution till you see the solution exit through the top filling port Click on the Close Fill Port button as soon as you see the liquid exit the top filling port The pipe...

Page 54: ...ette screw beyond repair The long needle and stir paddle on the pipette injection syringe must not be bent even slightly or baseline stability will be compromised Exercise care at all times in the han...

Page 55: ...librated Measuring large exothermic reactions will require a large reference power of about 30 uCal sec while large endothermic reactions will require a low reference power setting of about 2 uCal sec...

Page 56: ...d not be cleaned with detergent solution after each experiment since rinsing with water or buffer from a syringe is often adequate However after every 3 5 experiments or at the end of each day s exper...

Page 57: ...dapter see below Insert the long needle into the Sample cell and push down carefully until the o ring has sealed as shown below The end of the plastic tubing from the upper tube of the Cell cleaning A...

Page 58: ...ing After rinsing with water remove the plastic tubing from the rinse water and allow time for the vacuum to drain the fluid out of the plastic tubing then remove the cleaning apparatus from the cell...

Page 59: ...pply vacuum to the glass end of the injection syringe using the vacuum tube attachment for approximately 10 minutes to dry the methanol If it is necessary the cleaning brush included with accessories...

Page 60: ...imic a typical titration experiment with respect to the size of the heats Finally the Rnase 2 CMP provides a real experiment to be analyzed and compared with the results obtained with your instrument...

Page 61: ...or more information or suitable substitute This degassed water will be the solution that will go into the sample cell reference cell and syringe Fill the Cells Load the 2 5ml glass filling syringe wit...

Page 62: ...g needle of the injection syringe against any object as this could cause the needle to bend and expel some solution from the syringe and result in a poor first injection for the experiment If the long...

Page 63: ...e sample back into the filling test tube then raise the plunger to refill the injection syringe When the movement is complete the tip of the injection syringe will be positioned to its original positi...

Page 64: ...l and slowly lower the pipette When the pipette is almost completely inserted you may have to push down slightly to compensate for the resistance of the rubber o ring to seat the pipette Enter the Run...

Page 65: ...ive the query displayed below This is simply indicating that you have not entered a concentration This entry is irrelevant for the water experiment and you may select yes and continue If you were cond...

Page 66: ...showing the temperature rising to 30 degrees and stabilizing at the starting temperature Displayed above are three different Y axis scales with four different data sets being plotted in the graph Each...

Page 67: ...ss If this option was not selected the DP value that turned green becomes a button that will move the instrument to the next state when it is selected When VPViewer starts the Final Baseline Equilibra...

Page 68: ...periment The experiment will begin the DP display box will turn from green to black and the display title will read Pre Titration delay and then print the volume of solution that is left in the syring...

Page 69: ...ew 1 button 59 Using a y axis display range of 0 1 Cal sec And an x axis display range of 20 minutes 1200 sec your baseline should look similar to that shown above The slope and shape of the peaks may...

Page 70: ...ration Fill the sample cell with degassed water Prepare 20 ml of the degassed water in a test tube and add methanol 0 5 ml to make a 2 5 methanol solution Degas the methanol solution for 5 minutes whi...

Page 71: ...tically plots concentration normalized areas kcal per mole of injectant designated with an ndh extension into the Delta H template whereas what we want to view is the raw data heats cal per injection...

Page 72: ...grate all of your peaks one by one rather than rely on the automatic integration procedure and this will probably reduce scatter Again as a gauge of your success you may compare your results to the pl...

Page 73: ...analysis in Origin If your techniques are good you should be able now to generate the RNase 2 CMP binding isotherm and obtain fitting parameters very similar to those obtained at MicroCal with your i...

Page 74: ...is good practice to confirm the calibration every 3 months The Injection Syringe needle must be straight The Plunger Screw must be kept straight The syringe height i e position of the stir paddle from...

Page 75: ...aggressive cleaning agent after the others have failed Rinsing with water after using any cleaning agent is important WARNING The cells of the VP ITC are fabricated from Hastelloy C 276 and should nev...

Page 76: ...emove and dispose of the Contrad 70 A thorough rinse with distilled water should follow Cleaning Recommendations As a general rule the simplest effective method of cleaning is generally preferred Shor...

Page 77: ...eaning Agents Your choice of cleaning agents will vary with the cell contamination Below is a partial list of several agents and their applicability Top Job Mr Clean Is manufactured by Procter and Gam...

Page 78: ...ving old syringe plunger tip from pipette injector You must first separate the Syringe Holder from the pipette injector Place the pipette in its stand on the side of the instrument Open the Setup Main...

Page 79: ...the pipette screw out through the Guide Sleeve Occasionally the Tip Puller Tool will fail to successfully grab and remove the tip In this case the operator should move the plunger a little bit lower c...

Page 80: ...page Select the Install New Tip button Place a new plunger tip open end of plunger tip facing out into the end of the Tip Pusher Tool Insert the Tip Pusher Tool through the center of the Guide Sleeve...

Page 81: ...p the polishing aligned with the injector screw of the pipette As the tip is polished it will travel in the glass bore more freely and less force will be required to break it in A total of 30 to 50 st...

Page 82: ...t s original baseline position Entering a value for Pulse Spacing which is at least 4 minutes longer than the Pulse Duration will allow the deflected baseline to return to it s original value Users ar...

Page 83: ...An example of these results is shown below In general users can anticipate that the reported error in deflection or energy will be less than 1 If the error is reported as higher than 1 please call Mic...

Page 84: ...the pipette maintenance group click on the Remove Old Tip button Wait for the injector plunger to move up to its home position you will hear a beep when the movement is complete 4 Separate the injecto...

Page 85: ...of the syringe holder 14 Loosen the set screw of the upper Syringe Clamp so that the clamp is free to slide up and down on the outer diameter of the glass bore Lower the Syringe Clamp all the way down...

Page 86: ...ledge of the instrument and software it is strongly urged for the user to read through this section in its entirety before proceeding with experiments involving precious samples Please refer to our we...

Page 87: ...he intended run temperature The operator must also account for the change in temperature that will occur during the transfer of solution to the filling syringe and then into the cells If a solution wh...

Page 88: ...the cells and still be able to operate at the desired temperature the operator need only control the temperature of the solution to be loaded such that the final temperature of the cell after loading...

Page 89: ...ntrols group of the ITC Controls window to move the plunger ca 0 1 0 2 downwards The tip removal tool will then be able grab the plunger tip 6 3 A Note About Buffers DTT Dithiothreitol If possible DTT...

Page 90: ...ion of the binding component in the sample cell If Ka is unknown a best guess will have to suffice The molar concentration of the binding material in the syringe i e an enzyme inhibitor is estimated a...

Page 91: ...s Section 7 Additional Features This section contains information that the user may find useful but is not necessary for successful operation of the instrument This section is arranged as follows 81 7...

Page 92: ...to the jacket If you double click on this numeric value the display will change to a time function 7 2 Customizing VPViewer In instances of multiple users operating the same VP ITC system it may be ad...

Page 93: ...ialog box will open allowing you to change the current path Init Setup File Specifies the initial setup file that will be loaded into the experimental control window the first time it is opened Specif...

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