Please forward corrections or suggestions concerning this manual to info@MicroCalorimetry.com

Symbols

Symbols used in this manual
Warns the user of possible damage to the unit, draws attention to the risk of injury or contains safety notes and warnings.
Symbols used 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 Lab, Northampton, MA VP is a registered trademark of MicroCal, LLC, Northampton, MA MicroCal is a registered trademark of MicroCal, LLC, Northampton, MA Windows is a registered trademark of Microsoft Corporation

Operators Safety

The points below are intended to enhance your safety awareness and to draw your attention to risks which only you, the operator, can prevent. MicroCal has set high standards for ourselves in developing and constructing the VP-ITC. Each unit constructed is fully tested to operational and safety standards. It should be noted that no amount of design or constructed safeguards can anticipate or prevent improper handling. It is intended that this instrument be operated only by responsible people trained in basic laboratory protocol in procedures and safety techniques. This product is intended to operate from a power source that does not apply more than 260 V rms between the supply conductors or between either supply conductor and ground. A protective ground connection, by way of the grounding conductor in the power cord, is essential for safe operation To avoid any hazard, use only a fuse of the correct type, voltage rating and current rating as specified on the back of the VP-ITC. This is a 1 Amp 250 Volt Time-lag (Time Delay) fuse. Repairs, alterations or modifications must only be carried out by specialist personnel. The MicroCal Service Department will be happy to serve you for any repair work or operational questions. This device is not designed to the Medical Devices Directive 93/42/EEC and should not be used for medical purposes and/or in the diagnosis of patients. When using volatile or hazardous solutions in the cells, the operator 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 temperature before removing any solutions from the cells. All solutions in the cells must be cooled down below 50 C before removing from the cells to prevent the glass syringes from breaking due to the hot liquids.

Quantity ThermoVac Stir Bars (Small) Stir Bars (Medium) In-line filters
Test Tubes (Small) for conserving sample volume Test Tubes with Cap (Medium) Test Tube Holder Flask with Hose & Luer Lock Connector Fuses (1A/250V) 5x20mm Slo-Blo A.C. Mains Power Cord

Front Panel Controls

VACUUM
The ThermoVac is capable of pulling a maximum vacuum of 28.4 inches of mercury. If the VACUUM switch is pushed to the right the vacuum pump will turn on and remain on 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 (located at the rear of the instrument). When the switch is pushed to the left the vacuum pump will remain on till it is manually turned off (either by pushing the VACUUM switch to its middle, OFF, position or by turning off the main power switch).

TEMPERATURE

Use this dial to set the temperature, in C, for thermostatting the sample chamber. The rightmost digit sets the temperature value in tenths of a degree. Typically, ITC samples are thermostatted to a temperature that is below the desired run temperature for the sample. This should also allow for any temperature increase that may occur during the transfer of solution from the ThermoVac to the ITC cells or syringe.

SAMPLE TEMPERATURE

Displays the current temperature of the sample chamber.

STIRRER

This switch will activate a rotating magnetic field that will stir your sample when a small magnetic stir bar is placed in the tube containing your sample. You may adjust the speed of the Stirrer motor from 0 RPM (OFF) to the maximum speed of 800 RPM (full turn clockwise).
ADJUSTABLE VACUUM BLEEDER
Allows manual adjustment of the vacuum strength within the degassing chamber of the ThermoVac. There is no vacuum gauge on the ThermoVac, but the strength of the vacuum may need to be adjusted based on the behavior of your samples under vacuum. Depending on the specific sample being degassed, 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 strength of the vacuum with the bleeder screw. By turning the top of the bleeder screw in the counter-clockwise direction you will lower the strength of the vacuum within the ThermoVac and avoid boiling your sample. Similarly, the vacuum strength can be increased, by turning the top of the bleeder screw in the clockwise direction. The bleeder screw (and vacuum strength) can be fixed in a desirable position by tightening the lower nut, located at the base of the bleeder screw assembly, all the way in the clockwise direction.

Help. Allows access to the help menus. At the time of release of this Users Manual the on-line help was still under development. About. Provides specific details about this version of software and contact information for MicroCal, LLC.
VPViewer Main Window Buttons
The Main Window buttons allow access to VPViewers frequently used functions. The figure below shows all the buttons available for use. Depending on the current state of the instrument, some of the buttons would be grayed out and not available. (Specifically, the Start button is only available when the ITC is in its idle state. The Stop, Update Run Param. and Display Run Param. buttons are available when the VP-ITC is active, in a prerun, run or postrun state.)
Load Run File: Clicking on this button will display the file open dialog box to load previously saved run parameters into the ITC Controls Window. Run parameters may be loaded from one of two types of files; run parameters may be loaded from a previous experiments data file (*.itc), (the run parameters being extracted from the data file header) or they may be loaded from a setup file (*.inj) which was previously saved in the Save Run File option (as described below).
Save Run File. Displays the file save dialog box which allows saving of the currently displayed run parameters. By saving the run parameters to a setup file (*.inj) they may be reloaded and reused in the future by selecting Load Run File as described above Display Run Parameters. Displays the current run parameters for the run in progress. This button is available only when the VP-ITC is in a non-idle state. Update Run Parameters. Updates the current run parameters for the run in progress. When the ITC is in a non-idle state, this button must be clicked to realize the change of any run parameters. This button 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 stirring has commenced for an experiment, changes to the experimental stir speed will be ignored. Start. Click this button to start an ITC experiment, using the parameters entered into the ITC Controls Window. Stop. This button terminates the current run. Available only when the VP-ITC is in a non-idle state. Compact/Standard Mode Button Clicking this button will shrink VPViewers Main Window and change the icon to the Compact Mode (as shown below). VPViewers Main Window size cannot be manually adjusted. It has only 2 sizes, the compact mode size and the standard size. Clicking on the Standard Mode button will restore the window to its original size.

2.4 ITC Controls Window

The ITC Controls window (shown below) is the main access point for operating the VP-ITC instrument.
Below is a description for each of the run parameters displayed in the ITC Controls window.

Experimental Parameters

Total # Injections
Enter the total number of injections (500 is the maximum) for the titration (ITC) experiment.

Cell Temperature ( C)

Enter the desired temperature for the experiment. If there 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 current cell temperature. If there is no check mark, then the cell will scan to the entered temperature and equilibrate before the experiment will start. Please Note: When the instrument is programmed to scan to a new temperature (i.e. there is not a check mark next to the No Check Temp. option), the sample should be cooled (before loading into the cell) below the experimental temperature, so that after loading, the temperature of the sample in the cell will be
at or below the experimental temperature. If the loaded sample is at a higher temperature, there will be a long equilibration period before the experiment will begin. (See page 78 more information).
Reference Power (Cal/sec.)
Throughout an ITC experiment, a small constant amount of power (equal to the value entered here) is continuously supplied to the offset heater of the reference cell. This causes the DP feedback system to become positive to supply compensating power to the sample cell that will equilibrate the temperatures. During an experiment the DP baseline will equilibrate near the value entered in this box. The reference power setting is often referred to as the baseline setting. The best choice for the reference power setting will be determined by the anticipated size and direction of the titration peaks. Large exotherms will require a large reference power setting (ca. 30 uCal/sec.) and large endotherms will require a very small reference power setting (ca. 2 uCal/sec.).

Initial Delay (sec.)

Enter the time, in seconds, from the start of the experimental data collection till the time of the first injection. The minimum time allowed by VPViewer for this parameter is 60 seconds, this is necessary to establish a baseline prior to the first injection.

Volume (l)

Enter the volume of titrant (in micro liters), to be injected from the pipette into the sample cell for the selected injection(s).

Duration (sec.)

Enter the time, in seconds, for the titrant to be injected into the sample cell for the selected injection(s). Note: VPViewer will default this value to be twice the number entered in the Volume text box.

Spacing (sec.)

Enter the time, in seconds, between the beginning of the selected injection(s) and the beginning of the next injection. The user is responsible for specifying enough time between the injections to allow the DT signal to return 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 (sec.)

This is the time period (in seconds) in which the data channel conversions are averaged to produce a single data point for plotting and saving. For fast reactions, a filter period of 2 seconds is sufficient to obtain enough data points for representation of the peak for accurate integration of the area. For monitoring of very long, slow thermal processes, the filter period may be increased accordingly to avoid accumulation of excess data points. Please note: Although there may be a practical limit for a data set size, only available disk space would limit the number of data points that VPViewer is able to collect and save.

Edit Mode

All Same Selecting this option causes all injections to be assigned the same parameter values as entered into the Injection Parameter text boxes. Unique This option allows you to set up injections using different parameters. You may customize each injection to have different volumes, duration, spacing or filter periods. Editing the Injection Parameter text boxes when this option is selected effects only the injection that is currently highlighted in the summary table. Likewise the values displayed in the individual parameter boxes apply only to the injection that is currently highlighted in the summary table.
Apply To Rest Selecting this option, you will be assigning the injection parameters for the currently highlighted injection (in the summary table), to all ensuing injections. Likewise, editing an injection parameter while this option is selected affects the currently highlighted injection (in the summary table), as well as all ensuing injections.

Pipette Controls

This group of buttons control the injector movements of the pipette. The group box is labeled Ready when pipette controls are enabled or Busy when a movement is currently being made. The end of a movement is signaled by a beep and the group label, switching from Busy to Ready

Open Fill Port

This button is used for positioning the plunger tip for loading the pipette with your sample. When you click on this button the injector screw of the pipette plunger will retract till the plunger tip is positioned just above the fill port of the injection syringe. This will allow you to attach the hose of the plastic syringe to the fill port for filling the pipette injection syringe with your sample.

Close Fill 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 below the filling port of the injection syringe. This should be done immediately after filling the pipette syringe and before removing the hose of the filling syringe.

Purge->Refill

This button will completely extend the plunger tip of the pipette injector to inject the sample back into the filling test tube, then raise the plunger to refill the injection syringe. When the movement is complete the plunger tip of the injection syringe will be positioned to its original position, just below the filling port. A beep will indicate the start of the movement a second beep will indicate completion of the movement. The purpose of the purge/refill procedures is to dislodge any air bubbles from inside walls of the injection syringe (which may have occurred during the first filling) and expel them back into the titrant solution. It is recommended to perform this procedure twice, after loading the pipette before an experiment.

Distance (in.)

You may move the injector a set distance, either up or down, by entering the distance to be moved (in inches) in the text box and clicking either the Up or Dn (Down) button.
2.5 Thermostat/Calibration Window
The Thermostat/Calibration Window allows access to the thermostat control and DP calibration pulse function.

Thermostat Control

Thermostat Control allows for setting of the thermostat temperature, which will be maintained during the VP-ITC Thermostatting state. Pre-thermostatting the VP-ITC and samples will result in shorter equilibration times. Additionally, high temperature thermostatting during cell cleaning can often times improve the effects of the cleaning.

Y-Axis Scale

The Y-axis group allows for changing of the current y-axis (DP) units. Available y-axis units are mCal/min., Cal/sec. and Watts. By default, y-axis units will be displayed in Cal/sec. Changing y-axis units will only change the power units for local data displayed in real-time, within VPViewer and Origin. All data files will be saved with the default units of Cal/sec

Diagnostics

This group is used for diagnostics and troubleshooting and would normally not be utilized unless instructed by a MicroCal engineer. Export Current Data: Users may be instructed by a MicroCal engineer to click the Export Current Data button. This provides service personnel with a convenient means of capturing ITC data, which otherwise is not saved to disk. Plot Idle Data: Users may be instructed by a MicroCal engineer to checkmark the Plot Idle Data, for use in troubleshooting. This provides service personnel with a convenient means of capturing ITC data, which otherwise is not saved to disk. Extended Data Mode: This mode of operation will save all data the VP instrument produces to the data file. This provides MicroCal engineers with extra data for troubleshooting. Show Noise: When this box is checked the Real-Time Noise group box will be displayed. From this group you may elect to numerically display the RMS (Root-Mean-Squared) or PTP (Peak-To-Peak) noise.

Pipette Maintenance

This group of buttons provides controls for replacing plunger tips. It is recommended to replace tips once a month when using the VPITC daily. (For more information refer to the section on Replacing Pipette Plunger Tips starting on page 68). Remove Old Tip Click this button to position the injector plunger screw in a retracted position, so that the accessory 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 plunger tip on to the end of the plunger screw. Polish New Tip After installing a new tip, clicking this button will position the injector plunger tip (in an extended position) so that the glass polishing tool may be used to polish the plunger for first time use. New Tip Polished After polishing a new tip click this button to the plunger screw, this will position the screw properly for putting the pipette back together.

2.7 Constants Window

Provides 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 not be changed without instructions by a MicroCal Engineer.

PreRun Thermostat

Once the cells and jacket have reached the starting temperature, the ITC will enter the 2nd prerun state, the Prerun Thermostat state. The purpose of the PreRun Thermostat state is to maintain the starting temperature, for the requested period of time. This will allow time for thermal gradients within the ITC cell thermal core, to minimize before the experiment starts to reduce their affect during the experiment. The duration of the PreRun Thermostat state is 5 minutes.

PreStirring Equilbration

After the pre-run thermostat period has elapsed, VPViewer and the ITC cell will enter PreStirring equilibration. During this state the instrument will establish a zero degree DeltaT (temperature difference between the jacket and the cells) to establish adiabatic conditions suitable for sensitive thermal measurements. The DP signal will stabilize at a power level that is close (i.e within 1 cal/sec) to the Reference Power setting.
Final Baseline Equilibration
After the PreStirring Equilibration has been reached, the VP ITC instrument will enter the Final Baseline Equilibration or the stirring state. The VP will start rotating 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 instrument will stir continuously for the remaining portion of the run.

Running

Once the DP signal has stabilized after the start of the stirring, the instrument will enter the run state. The run commences with an initial delay to provide baseline data prior to the first injection. Then the injections will be executed according to the injection schedule. The instrument responds to an exothermic reaction by decreasing the feed back heat provided to the sample cell. That is, heat added to the sample cell by an exothermic reaction will result in a downward deflection in the baseline DP signal. This downward deflection is proportional to the energy not needed to maintain the temperature equilibrium between the sample and reference cells. The integrated area of this deflection is referenced to the baseline before the injection and after the reaction has completed. An endothermic reaction will require more heat in the feed back loop to maintain the same temperature resulting in a positive deflection.

The end of the plastic tubing from the upper tube of the Cell cleaning Apparatus is immersed into a beaker of 200-400 ml of detergent cleaning solution. The end of the plastic tubing from lower tube is connected to a one liter vacuum flask through the #8 rubber stopper. The side arm of the vacuum flask is attached to the Vacuum Port of the ThermoVac. Your Setup up should look like below.
Turn on the ThermoVac vacuum pump. The vacuum will pull the detergent solution from the beaker, through the cell and into the waste flask. NOTE: DO NOT ALLOW THE VOLUME OF FLUID IN THE WASTE FLASK TO BECOME SUCH THAT IT WOULD RISE UP TO THE LEVEL OF THE SIDE ARM AND BE SUCKED INTO THE THERMOVACS VACUUM PUMP. THIS MAY CAUSE THE PUMP TO BECOME DAMAGED. Once sufficient detergent solution has passed through the cell, the plastic tubing is removed from the solution, rinsed free of detergent using a plastic wash bottle, and then placed into another beaker containing 300 ml or more of water for rinsing. 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. Remove the remaining water from the cell by using a long needle syringe.
We do not recommend drying the cell before filling with your sample solution, but it should be rinsed twice with the buffer you are using for the experiment. Finally, it should be completely drained and filled with your sample solution in the manner described earlier. Because a small amount of the buffer used for final rinsing will adhere to the walls of the cell and act to dilute your sample solution, you may wish to correct for this by lowering your sample concentration by 2% if you measured concentration before the sample was introduced into the cell.

3.8 Cleaning Syringes.

Cleaning Injection Syringes
Disconnect the syringe from the pipette and insert the accessory plunger into the top of the syringe. Attach the plastic loading syringe tube to the filling port at the top of the syringe Insert the tip of the injection syringe into the cleaning solution and draw 2ml of solution through the syringe Empty the plastic loading syringe and repeat the above step at least two more times. Insert the tip of the injection syringe into distilled water and draw 2ml of solution through the syringe Empty the plastic loading syringe and repeat the above step at least two more times. Insert the tip of the injection syringe into HPLC Grade methanol and draw 2ml of solution through the syringe. Empty the plastic loading syringe and reattach it to the port. Remove the accessory plunger. Reattach the plastic loading syringe. Apply vacuum to the glass end of the injection syringe using the vacuum tube attachment for approximately 10 minutes to dry the methanol.

has equilibrated in the Pre-stirring mode you may want a closer look at the data, so click on the Auto-View 1 button. This will put the current data point at the center of the graph with a Y-axis full scale of 0.1. You may notice that when the graph first appears in Origin, the DP data display box in VPViewers main window has the values displayed in red. Once VPViewer determines that the cell has equilibrated in the Pre-stirring mode, the data display will turn to green. This means that the cell is ready to enter the stirring mode of equilibration. Since Auto Start in the ITC Equilibration Options group was enabled in the ITC Controls window, VPViewer will automatically start the stirring and apply power to the reference cell, moving on to the next state of the equilibration process. 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 Equilibration it starts the stirring and applies power to the reference cell. The amount of power applied to the reference cell is determined by what you entered into the Baseline Position (uCal/sec) text box located in ITC Controls window. For this tutorial we entered 5 for the Baseline Position, you may see that there may be an initial decrease in the DP power (due to the added frictional energy in stirring), but then the DP values will increase because of the power applied to the reference which forces the feedback system to apply power to the sample cell to compensate and maintain a temperature balance. The effect of this heat disturbance will take a few minutes for a new equilibration level with the final DP values being close to 5. Select the Rescale to Show All button and you will see a graph similar to the above.
You may want a closer look at the final baseline before proceeding. Click on the Auto-View 1 button. You should see a view similar to that below.
When VPViewer determines that the Final Baseline has equilibrated properly, the display in the DP box will turn from red to green. Since Auto was checked in the ITC Equilibration Options group, VPViewer will automatically start the experiment. (If the automatic mode was disabled you would now need to double-click anywhere in the DP display box, to start the experiment). 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 syringe. The Pre-Titration Delay is required for an initial baseline for the first injection of the experiment. The time period for this initial baseline was entered in the Initial Delay text box located in the ITC Equilibration Options group of the ITC Controls window.

Once the menu has been selected, the Calibration Pulse Setup Window will appear. This window will serve as the pulse parameter editing window where users will define the calibration pulses to be entered. Individual pulse parameters are entered by first selecting a pulse or multiple pulses, then entering the desired parameter value into the pulse parameter boxes (Calibration Power, Pulse Duration and Pulse spacing). This window is pictured below:
The parameters shown in the above window will allow for setting of all relevant y-axis calibration parameters with the exception of the delay period prior to the first pulse. This variable
comes from the Standard ITC setup menu as the Initial Delay value. This value should be at least 60 seconds. Users are encouraged to simply use the default y-axis calibration parameters. After the run and pulse parameters are entered, clicking on the Start Run button will start the run. The ITC will equilibrate in the same manner as it would during a titration experiment. After the final equilibration phase has completed, the initial delay will begin and the pulses will be applied as entered. As each pulse completes, VPViewer will analyze the pulse region and determine the deflection of the baseline as well as the energy (area) of the pulse. The requested power and energy will also be displayed as will a percent error for both power and energy. It is sufficient for users to gauge the accuracy of their y-axis calibration by the results supplied by VPViewer, in the script window. 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 MicroCal and speak to a Service Representative. If a more thorough analysis of y-axis calibration results is desired, the calibration data file can be read into Origin just as a titration data file can for detailed analysis. Also, it is important that users realize that the data displayed in VPViewer, in real time, will change in its appearance after each pulse analysis is carried out. After each pulse is turned off and the DP signal has re-equilibrated to its original baseline, the data is operated on within VPViewer. The pulse region will have a linear baseline subtracted from it resulting in the baseline portion of the pulse region to be offset very close to zero. After this subtraction it is easy to gauge the actual deflection of the pulses from the graph. Also, it need be mentioned that only the data within VPViewer will be operated on in this way and that the *.itk data file will contain only raw data.

11. Lower the upper Syringe Clamp so that it is flush to the top of the syringe holder. 12. Tighten the set-screw of the upper Syringe Clamp so that the disk is fixed to the syringe glass. 13. Raise the syringe by carefully prying up on the upper Syringe Clamp until it is approximately 3 mm higher than the top 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 so that it is again flush to the top of the syringe holder. 15. Tighten the set-screw of the upper Syringe Clamp and your syringe height is now fixed at 3 mm from the bottom of the sample cell.

Reassemble the Pipette

Please refer to Section 1.6 Pipette Assembly (page 9), for instructions on how to reassemble the pipette with the new syringe
Section 6: Tips and Troubleshooting
This section provides tips for optimizing the performance of the ITC instrument and troubleshooting techniques for commonly encountered problems. After a new user has gained a working knowledge 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 web site at www.microcalorimetry.com for updated tips, troubleshooting and application notes.
This section is arranged as follows: Section 6: Tips and Troubleshooting....76

6.1 User Tips and FAQs

1.) When powering up the VP-ITC, always follow the sequence listed below: 1. Turn on PC power 2. Turn on VP-ITC power 3. Launch VPViewer 2.) After initial power up of the VP-ITC, the data channels will display incorrect values until the power LED on the front of the cell unit becomes lit. Once the power indicator light comes on, all data channel readings will be accurate and the instrument is ready to use. 3.) When the VP-ITC requires cooling to get to a target temperature (thermostat or run temperature) it uses an algorithm which will do the following: - Set shield to target minus 10 degrees to help cool jacket/cells via conduction/convection. - Wait for jacket temperature to be cooler than target minus five degrees. - Set shield to target minus three degrees to bring jacket and cells closer in temperature. - Wait for deltaT to be non-saturated (small jacket-cell deltaT). - Set shield to target minus one degree. - Heat cells and jacket to target While this algorithm requires more time than heating, it will cool the VP-ITC to the desired temperature as fast as possible and the operator should not interrupt the algorithm in efforts to speed up the cooling process. For very low temperature operation of the VP-ITC (less than 10 degrees), users should call MicroCal for consultation. 4.) When filling the cells it is important to load solution that is colder than the 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 which is hotter than the ensuing run temperature is loaded into the cells, the ITC will likely require cooling in order to get a precise starting temperature for the run. Alternatively, the operator could check the No Check T option in the ITC setup window. This will tell VPViewer to allow the jacket to be heated up to the cell temperature and commence with the pre-run equilibration. 5.) When filling the injection syringe, there are a couple of things that should be avoided in order to get the optimum performance form the ITC. - Do not allow the syringe needle or paddle to be flexed when drawing solution into the syringe during filling. During syringe loading this can sometimes occur if the sample tube is not straight in the threaded tube holder, or if the threads themselves were not mated correctly. If you can see that the needle or paddle is hitting the side or bottom of the sample tube, correct the situation before you actually draw your solution into the syringe. - Do not allow the solution level to go below the bottom tip of the paddle when drawing your titrant into the syringe. Be sure to close off the flow path (close port button) before the solution level gets too low. The sample tube can be positioned in its holder to adjust its height relative to the bottom of the paddle if necessary. - Always avoid flexing of the injection syringe needle when inserting and removing it from the cell.

6.2 Troubleshooting

PROBLEM: Long equilibration period before an experiment starts. There is no capability for directly cooling the cells nor the inner jacket of the VP-ITC, as was also the case for our earlier ITC instruments. If the operator is carrying out a series of experiments in the VP-ITC at 25.0 C, for example, and if the freshly loaded sample solution has a temperature of 26.0 C, then there are but two choices. If the No Check T option in the set-up window is checked X and the experiment started, then the system will equilibrate quickly at 26.0 C. If the No Check T option is unchecked then the software begins a process to cool the cells. This process consists of first lowering the temperature of the outer shield (which has cooling capability) far below 25 C which in time acts to lower the temperature of the inner jacket and ultimately the cells, both of which must cool by heat transfer to the outer shield. After the cells and inner jacket have cooled to 25 C, the temperature of the outer shield is cycled back up to its set position to begin the experiment. Because heat exchange with the outer shield is slow, this process can take a significant amount of time, even when the cell temperature only needs to be decreased by a degree or so.
AVOIDING THE PROBLEM: To avoid the lengthy delay in cooling 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, is at or below the temperature where the experiment will be carried out. When this is the case, the No Check T option should be left unchecked and the cells will promptly be heated to the correct temperature (without changing the temperature of the outer shield) so that the experiment will be ready to begin in a short time. For experiments below room temperature, solutions should initially be cooled (after degassing) significantly below the set temperature since they will warm up somewhat when drawn into the loading syringe and transferred into the cell.
PROBLEM: Baseline is stable without stirring, but becomes unstable after syringe is inserted and stirring begins prior to injections. SOLUTION: Indicates a stirring problem. May be debris or bubbles in cell, bent injection syringe needle, improper syringe height adjustment, or injection system alignment problem. Debris or bubbles refill cell after rinsing or using cell cleaning apparatus and try again. Extraordinary circumstances may require carefully inverting the entire VP-ITC unit on a suitable stand and flushing the sample cell with the filling syringe to remove solid debris. If your test samples are suspensions of particles try higher stirring rate to achieve uniform suspension, although the extra noise caused by the increased stirring speed may cause deterioration of the baseline.

Please forward corrections or suggestions concerning this manual to info@MicroCalorimetry.com

Symbols

Symbols used in this manual
Warns the user of possible damage to the unit, draws attention to the risk of injury or contains safety notes and warnings.
Symbols used 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 Lab, Northampton, MA VP is a registered trademark of MicroCal, LLC, Northampton, MA MicroCal is a registered trademark of MicroCal, LLC, Northampton, MA Windows is a registered trademark of Microsoft Corporation

Operators Safety !

The points below are intended to enhance your safety awareness and to draw your attention to risks which only you, the operator, can prevent. MicroCal has set high standards for ourselves in developing and constructing the VP-ITC. Each unit constructed is fully tested to operational and safety standards. It should be noted that no amount of design or constructed safeguards can anticipate or prevent improper handling. It is intended that only responsible people trained in basic laboratory protocol in procedures and safety techniques operate this instrument. This product is intended to operate from a power source that does not apply more than 260 V rms between the supply conductors or between either supply conductor and ground. A protective ground connection, by way of the grounding conductor in the power cord, is essential for safe operation To avoid any hazard, use only a fuse of the correct type, voltage rating and current rating as specified on the back of the VP-ITC. This is a 1 Amp 250 Volt Time-lag (Time Delay) fuse. Repairs, alterations or modifications must only be carried out by specialist personnel. The MicroCal Service Department will be happy to serve you for any repair work or operational questions. To enhance safety always plug the instrument into a Ground Fault Circuit Interrupter (GFCI) device. A solution can become an electrical conductor when in contact with electricity and can create a hazard with the potential of burns or death. Use caution when using solutions near the instrument and adhere to the following items:

! ! ! !

! ! ! ! !
If any liquid is spilled on or around the instrument unplug the instrument immediately and wipe it up, if there is any possibility that some of the liquid may have leaked into the instrument case contact MicroCal immediately. DO NOT PLUG THE INSTRUMENT INTO ANY POWER MAINS, until the problem is resolved. Do not over fill the cell and reservoir. There is a small reservoir located at the top of the access tubes to catch a minimum amount of overflow, do not exceed the capacity of the reservoir and allow any fluid to leak into the instrument cabinet.

Instrument Safety Compliance
MicroCal VP-ITC calorimeters carry the CUE Safety Certification Mark, authorized by TV America, a division of TV Sddeutschland, to signify that: 1) the instrument has been tested by an accredited Certification Body and meets applicable Canadian electrical safety standards/requirements (CSA/SCC). 2) the instrument has been tested by an NRTL (Nationally Recognized Testing Laboratory) and meets applicable United States electrical safety standards/requirements (ANSI/UL). 3) the instrument has been tested by a Competent and Notified Body for applicable EU Directives and meets applicable safety standards/requirements (EN/IEC).
Section 1: VP-ITC System Introduction
This section starts by providing some basic information about the VP-ITC and the computer controller. Then it provides instructions for setting up the VP-ITC instrument and accessories. It ends by providing information about the ThermoVac instrument.

1.1 VP ITC The Basics

The VP-ITC (Isothermal Titration Calorimeter) Unit directly measures heat evolved or absorbed in liquid samples as a result of mixing precise amounts of reactants. A spinning syringe is utilized for injecting and subsequent mixing of reactants. Spin rates are user selectable. The normal operating range is 2C to 80C. Wetted cell surfaces are Hastelloy Alloy C 276. Sample and reference cells are accessible for filling and cleaning through the top of the unit. The sample cell is on the right as one faces the front of the unit. A pair of identical coin shaped cells is enclosed in an adiabatic Outer Shield (Jacket). Access stems travel from the top exterior of the instrument to the cells. Both the coin shaped cells and the access stems are totally filled with liquid during operation. This requires approximately 1.8 ml. per cell even though the working volume of the cell is only 1.4 ml Temperature differences between the reference cell and the sample cell are measured, calibrated to power units and displayed to the user as well as saved to disk. The data channel is referred to as the DP signal, or the differential power between the reference cell and the 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 of power through a resistive heater element located on the cell. The syringe containing a ligand is titrated (injected) into the cell containing a solution of the macromolecule. An injection which results in the evolution of heat (exothermic) within the sample cell causes a negative change in the DP power since the heat evolved chemically provides heat that the DP feedback is no longer required to provide. The opposite is true for endothermic reactions. Since the DP has units of power, the time integral of the peak yields a measurement of thermal energy, H. This heat is released or absorbed in direct proportion to the amount of binding that occurs. When the macromolecule in the cell becomes saturated with added ligand, the heat signal diminishes until only the background heat of dilution is observed. With the VP-ITC system the entire experiment takes place under computer control. The user inputs the experimental parameters (temperature, number of injections, injection volumes) and the computer carries out the experiment. Origin software is then used to analyze the ITC data using fitting models to calculate reaction stoichiometry (n), binding constant (Kb), enthalpy (H) and entropy (S).

2.3 VPViewer Main Window

When you launch 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

In the above illustration, the instrument is Thermostatting (ITC) at 30 C.

Temp(Celsius)

This text box displays the current temperature, in degrees Celsius, of the adiabatic jacket.

DP (uCal/sec)

This text box displays the Differential Power, in Cal/sec., between the reference cell and the sample cell. Positive DP values means that the reference cell is hotter than the sample cell, while negative DP values means that the sample cell is hotter than the reference cell. In terms of reactions (assuming they take place in the sample cell) exothermic reactions will cause the DP to deflect in the negative direction while endothermic reactions will cause the DP to deflect in the positive direction. The power units for the DP data channel may be displayed in mCal/min., Cal/sec. or Watts. Experimental data will always be saved in units of Cal/sec. Whenever the VP-ITC is equilibrated the DP value should be positive and stable. The power level of the equilibrated DP signal should be close (+/- 1 Cal/sec.) to the Reference Power setting, as set prior to starting the run.

DT (deg. C)

This text box displays the Differential Temperature, in degrees Celsius, between the adiabatic jacket and the calorimeter cells (i.e. the average temperature of both cells). Whenever the VP-ITC is equilibrated the DT signal should be close to zero degrees Celsius (+/0.05) and stable.

Menus and Buttons

The VPViewer Main Window contains drop down menus, buttons and Window Tabs. The Main Drop Down Menu selections generally control global system functions The Main Window Buttons allow easy access to features that are frequently used. The Main Window Tab Selections provide detailed windows for specific functions. The details of these controls can be found in the following sections.
VPViewer Main Drop Down Menus
The Main Drop Down Menus at the top of VPViewer will provide access to some of the less frequently used functions of the application.
System: Quit Program. Selecting Quit Program prompts the user to see if they want to terminate the VPViewer application. Responding with yes will terminate the program and any run in progress. All ITC run, or partial run data will be saved to disk. Approximately a minute or so after program termination, the power to the VP-ITC will also be shut down. Power to the VP-ITC will not be restored until the VPViewer application is run again.

The Setup/Maintenance window allows access to change user preferences for the default settings of run parameters. Systems having multiple users may benefit from having customized settings.

User Setup

Data File Path: Indicates the path (excluding file name) where VP-ITC data files will be saved. The path should be located on a local hard drive and not on any floppy or CD-ROM drive. This setting may only be changed while the VP-ITC is in the idle state, Thermostatting. To change 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 from the dialog box. Note: In order to be read into Origin for data analysis the path cannot contain any spaces. Setup File Path: Indicates the path (excluding file name) where VP-ITC setup files will be saved and loaded. The specified path should be located on a local hard drive and not on any floppy or CD-ROM drives. To change the current setup file path, double click on the existing path text and a dialog box will open allowing you to change the path. The desired setup directory must already exist in order to select it from the dialog box.
Init. Setup File: Specifies the initial setup file, which will be loaded into the experimental control window, the first time it is opened. Specifying the initial setup file as LastRun1.inj will result in the last run parameters that were executed to be loaded at startup. Cells Boot Temperature: Upon launching VPViewer, the ITC will scan to, and then thermostat at, the temperature entered into this text box. Default Shield DT: The default temperature difference between the ITC adiabatic jacket and the outer shield of the VP-ITC. The default setting of a 1 degree difference has been found to be ideal for all ITC applications and should not be changed without consultation with a MicroCal engineer. Analog Input Range: Determines the maximum range of measurable DC voltages for all VPITC data channels. The default range of +/- 1.25 volts is adequate for almost all ITC applications and provides for the greatest sensitivity. In instances where heats are too large to be measured using the default setting, increasing the Analog Input Range can increase the range of DP measurement. Please consult a MicroCal engineer prior to making this change. Save/Add/Erase User: These buttons provide a convenient way of maintaining unique startup parameters and placing data files in different folders for multiple users. (See section 7.2 Customizing VPViewer, starting on page 89, for more information)

Click OK to exit and save any changes or Cancel to exit without saving any changes.

VP-ITC Numeric Display

This group of display text boxes shows the same data channels as VPViewer, as described in section 2.3 VPViewer Main Window.

2.9 ITC Cell Status

States of Operation
The VP-ITC is a state driven instrument. The current state of the ITC (cell status) is displayed in both VPViewer and Origin. There are six unique states that comprise an ITC run, thermostatting (idle), Seeking Temperature, PreRun Thermostat, Prestirring Equilibration, Final Baseline Equilibration and Running. A description of each of these six states can be found below.
Thermostatting (idle state)
When the VP-ITC is not equilibrating or performing a titration run, it sits in an idle state, the Thermostatting State. While in the thermostatting state, the only function for the VP-ITC is to achieve and maintain the currently defined thermostat temperature. The VP-ITC will remain in its idle state until the operator starts a run. Access to the current thermostat temperature is obtained through the Thermostat/Calib. Main Window tab.
Seeking Temperature (Heating/Cooling Jacket/Cells to Set Temperature)
The function of this first prerun state is to heat or cool the VP-ITC cells and adiabatic jacket to the entered starting temperature for the experimental run. This is 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 equilibration time it is usually best to set the VP-ITC thermostat temperature to be the same as the starting temperature of the ensuing run, or colder. When filling the cells with solution prior to a run, the sample being loaded should always be colder than the desired experimental temperature, in order to minimize the amount of time required to equilibrate the ITC. Loading solution that is hotter than the desired run temperature will cause for lengthy equilibrations and/or a run temperature that is slightly higher than requested. When the VP-ITC requires cooling to achieve the requested starting (or thermostat) temperature, it uses a different algorithm than when it requires heating. Since there is no active cooling of the cells or jacket, the outer shield will be used to pull the jacket and cells lower in temperature through conduction and convection inside the ITC. To increase the rate of cooling, the outer shield will be cooled well below (10 degrees below) the requested target temperature, causing the jacket to go well below the target temperature. Although the jacket and outer shield will be well below the target temperature, the cells will take longer to cool and the algorithm will wait for the cells to be below the target temperature. Once the jacket and cells are below the target temperature they will be heated up to the requested target temperature.

c = KMtotn

Very large c values lead to very tight binding and the isotherm is rectangular in shape with the height corresponding exactly to Ho and with the sharp drop occurring precisely at the stoichiometric equivalence point n in the molar ratio Xtot/Mtot. The shape of this curve is invariant with changes in K so long as the c value remains above ca. 5000. As c is reduced 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 limit of very low initial Mtot concentration (cf., c=0.1), the isotherm becomes featureless and traces a nearly horizontal line indicative of very weak binding. It is apparent from looking at these isotherms that their shape is reasonably sensitive to binding constant only for c values in the range 1 < c < 1000, corresponding to binding of intermediate strength. We will refer to this range as the "experimental K window". When available, the middle of the window from c = 5 to 500 is most ideal for measuring K.
The correct choice of macromolecule concentrations for an experiment depends both on the objective of the experiment (i.e., whether you wish to determine a Ho of binding only, or whether you wish to determine n and K in addition to Ho) and upon the magnitude of K. While considering your choice of starting concentration, it must also be remembered that the limiting
VP-ITC sensitivity is ca. 0.1 cal so for precise measurement each injection should have an average of at least 3-5 Cal of heat absorbed or evolved into the 1.3 ml cell. How these factors impinge on your choice of macromolecule concentration can be seen by considering a particular example of the binding of 2'CMP to ribonuclease A, where the binding constant is approximately 1 x 106 M-1 and the Ho is approximately -15000 cal/mole for the single binding site.
A. Measuring Ho, K and n by deconvolution of total binding isotherm.

For a K of 106, RNASE concentrations are in the experimental K window for the Molar range 106 < M < 10-3. It requires at least 10 separate injections to define the total binding isotherms tot and each injection must average ca. 5 cal, so the total heat Q required in the 1.3 ml cell is 50 cal, i.e., Q = 50 x 10-6 cal = (15000 cal/mole) (Mtot moles/l ) (1.3 x 10-3 l ) Solving this equation for Mtot gives a minimum concentration of ca. 3 x 10-6 M. This concentration is larger than the lowest concentration, 1 x 10-6 , in the experimental 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 choose concentrations higher than the minimum of 3 x 10-6 so that Q signals will be larger and c values will be in the ideal range between 10 and 100. (i.e., 10-5 < Mtot < 10-4 ).
B. Measuring only Ho by single ligand injection into excess macromolecule.
To measure Ho by a single injection (i.e., without deconvolution of the total binding isotherm) requires a c value large enough so the experimental intercept on the isotherm intercepts the Y axis very close to the true Ho, i.e., c > 100. This means Mtot > 10-4. Since there will be excess macromolecule in the cell, the experimental heat Q will be determined by the amount of ligand injected, i.e., Q = (15,000 cal/mole) (syringe conc.) (inj. vol.) For example, a 10 ml injection of a 7 x 10-5 M ligand solution would give the minimal 10 cal of heat. It is also possible to measure H by injecting excess ligand into a very low concentration of macromolecule. Referring to case A above, once you have chosen Mtot you must select the ligand concentration Xtot for the solution to be loaded into the syringe. This will depend on the volume of the syringe
that you plan to use. For c values larger than ca. 10, the final concentration of ligand in the cell after all injections are completed should be ca. 1.5 times the total concentration of macromolecule binding sites in the cell at the beginning of the experiment,
i.e., Xtot x v/V = n x Mtot x 1.5,
Where v is the total volume of injectant to be used, V is the cell volume (ca. 1.3 ml), and n is the ligand/macromolecule stoichiometry. For cases where n=1, the ligand concentration, Xtot,, should be ca. 8.5 times Mtot using a 250 l syringe. If the c value for your system is lower than 10, you may wish to increase the final ligand/macromolecule ratio from 1.5 up to 2.0 or even 2.5 as is evident by referring back to the previous figure showing binding isotherms as they depend on c. It should be realized however that accurate curve fitting is possible even when saturation of sites is not achieved. There may be other factors, specific to your system, that are important considerations in experiment design, such as the total amount of macromolecule or ligand that is available for the experiment and/or solubility restrictions on the macromolecule or the ligand. Several other experimental design problems should be mentioned. First, the buffer in which the ligand is dissolved should be an exact match (i.e., pH, buffer concentration, salt concentration, etc.) to the buffer in which the macromolecule is dissolved, or else large spurious heat effects from buffer mixing will result. For example, if the ligand is dissolved directly into the buffer which was dialyzed against the macromolecule solution, the exact pH of the ligand solution may 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 before doing the experiment. Second, control experiments (i.e., ligand solution added to buffer in cell without the presence of macromolecule) to determine the heat of dilution of ligand should be carried out in the same way as the experiment with macromolecule present, and these heats of dilution should be subtracted from the corresponding injection into the macromolecule solution. You will usually find these heats of dilution to be small and frequently negligible (unless the ligand dimerizes or aggregates with itself!) but they should be checked as a precaution. Finally, you may find occasionally that the first injection in a series of injections shows a smaller heat effect than it should. This can result from bending the syringe needle a little when seating the injector into the barrel, or leakage resulting from having the syringe in the cell a long time before the first injection is made (particularly if it is stirring all the while). It you find this to be a persistent problem with certain systems, even when care is taken to avoid the aforementioned factors, you may wish to make a small first injection (e.g. one 1 l injection followed by ten 10 l injections) and then delete the first data point before doing curve-fitting in Origin. Because of release or uptake of protons during many biological binding reactions, the observed heats of binding may be strongly dependent on which buffer is used. In fact, certain binding reactions which have extremely small H and produce virtually no signal in buffers with small Hion (e.g., phosphate) can sometimes be studied nicely in buffers with a large Hion (e.g., tris) where the signal will be much larger.

3.2 Using the ThermoVac to Degas Samples
Degas cell and syringe samples that may contain dissolved gas to insure bubble free loading of each. This is particularly important if samples recently were at refrigerator temperatures. A ThermoVac sample degassing and Thermostat station is provided with each instrument. The ThermoVacs preset vacuum time of ca. 5 minutes is adequate to degas a sample that is being stirred, but a much longer time is required without stirring. Normally, you would set the ThermoVac temperature to 1 to 5 degrees below the intended experimental temperature while degassing. If volatile buffers or ligands are being used then solutions should be prepared from degassed or pre-boiled water and stored air-free. If sample solutions contain any undissolved solutes or extraneous solid material of other types, they should be filtered before 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 will prevent a long equilibration period before the experiment can start (see page 87 for more information). Therefore, if running an experiment much below room temperature you may wish to set the ThermoVac temperature 5 degrees below the experimental temperature, as the sample may warm up during the transfer to the calorimetric cells. Additionally, the temperature of the filling syringe itself will act to change the sample temperature during cell filling. For this reason users may consider using a cold syringe (stored in a refrigerator) to minimize the sample temperature change during cell filling. The temperature of the injection syringe sample has little effect on the equilibration period.

Top View of ThermoVac

Note: The ThermoVac includes a preinstalled bleeder valve on the top of the vacuum cap to allow manual adjustment of the vacuum strength in the degassing chamber. If the vacuum is
boiling the sample in the chamber you should adjust the adjusting knob of the bleeder valve counterclockwise to reduce the vacuum. Any customers with a ThermoVac that does not include this option may request one through our sales department. To degas solutions before placing into the cells or injection syringe, please do the following. Turn on the Power Main Switch. Set the desired temperature for the solution. Place your solution to be degassed into a test tube, add a small stir bar and place the tube into one of the open cylinders of the Tube Holder insert. Please Note: To conserve titrant sample for loading into the injection syringe, use the small test tubes provided with the ThermoVac. If you wish to use a tube or beaker larger than will fit into the Tube Holder you may remove the Tube Holder by simply lifting it up. Due to the tight fit of the Tube Holder, it may be difficult to lift the Tube Holder out of the sample chamber, in this case you may use a 3/32 hex (Allen Key) wrench to turn a screw , located at the bottom of the center hole of the Tube Holder, to lift the Tube Holder out. Turn the stirring on. Turn on the vacuum. Push the switch to the right to activate the vacuum for a preset (ca. 5 minutes) duration. Push the switch to the left if you wish to manually control the time for the vacuum. If excessive bubbling occurs that could boil over your sample you may adjust the bleeder valve. Turning the adjusting knob clockwise will increase the vacuum while a counterclockwise turn will decrease the vacuum strength. Place the Vacuum cap on top of the sealing o-ring. The sound of the vacuum pump will change pitch to indicate the vacuum has sealed the Cap to the o-ring. Once the vacuum has sealed, the Vacuum Cap will be held firmly in place, until the vacuum pump shuts off. Please Note: Once the vacuum pump has been turned off, the vacuum in the sample degassing chamber may be released at any time causing the Vacuum Cap to fall off the sealing o-ring. Make sure that there is nothing on top or next to the ThermoVac that may spill or break due to the sudden movement of the Vacuum Cap.

10.0 9.5 9.0

Conventional ITC Binding Experiment

29.5 29.0 28.5 28.0

Single Injection Method ITC Binding Experiment

D P (uC al/sec.)

8.0 7.5 7.0 6.5 6.0 5.10000

D P (uC a l/sec.)

27.5 27.0 26.5 26.0 25.5 25.0 24.500 1000

Time (seconds)

Single Injection Method ITC Binding Experiment Conventional ITC Binding Experiment one large injection multiple, discrete injections produces binding isotherm in real-time reequilibration to baseline after each injection total time of experiment approximately 1 hour or more total time of experiment approximately 20 minutes

Activating the SIM Mode

The SIM mode of operation is activated in VPViewer 2000 under the ITC/SIM Mode menu. Select this menu and the ITC controls window will change to the SIM mode. The SIM Mode menu will display a check mark when the SIM mode is active. The other indication that you are in SIM mode is the Total # Injections parameter will be set to 1 and is grayed and unchangeable. Additionally, the data file extension for all SIM data files will be.sim. To switch back to the conventional ITC mode, again select the ITC/SIM Mode menu. The check mark next to the menu will be removed and the VPViewer 2000 software will be switched back to conventional ITC mode.
The SIM Mode Run Parameter Window
The ITC run parameter window will automatically display default values, appropriate for the SIM mode of operation, when the SIM mode is enabled. The values that are significantly different from the standard ITC mode are stirring speed, injection volume, injection duration, injection spacing and filter period. The SIM injection volume is much larger than with traditional ITC experiments, and may be as large as the entire syringe volume (ca. 295L). The injection duration is automatically set to a value that is 10 times larger than the entered injection volume which results in an injection rate of approximately 100nL/sec. The injection spacing must exceed the injection duration by at least 300 seconds to allow complete reaction of the titrant and ligand. The sum of the injection spacing and the initial delay period will determine the total duration of the experiment (not including the pre-run equilibration period). A default filter period of 2 seconds is a good choice and will rarely need to be changed. A picture of the ITC run parameter window, in SIM mode, is shown below.

3.8 Using the ThermoVac for Cleaning the ITC Sample Cell
The ThermoVac may be used to flush copious amounts of detergent solution followed by a thorough rinse with distilled water. Insert the Cell Cleaning Apparatus into the Adapter, until the top flange is touching the top of the Adapter (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 Apparatus is immersed into a beaker of 200-400 ml of detergent cleaning solution. The end of the plastic tubing from lower tube is connected to a one liter vacuum flask through the #8 rubber stopper. The side arm of the vacuum flask is attached to the Vacuum Port of the ThermoVac. Your Setup up should look like below.
Turn on the ThermoVac vacuum pump. The vacuum will pull the detergent solution from the beaker, through the cell and into the waste flask. NOTE: DO NOT ALLOW THE VOLUME OF FLUID IN THE WASTE FLASK TO BECOME SUCH THAT IT WOULD RISE UP TO THE LEVEL OF THE SIDE ARM AND BE SUCKED INTO THE THERMOVACS VACUUM PUMP. THIS MAY CAUSE THE PUMP TO BECOME DAMAGED. Once sufficient detergent solution has passed through the cell, the plastic tubing is removed from the solution, rinsed free of detergent using a plastic wash bottle, and then placed into another beaker containing 300 ml or more of water for rinsing. 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. Remove the remaining water from the cell by using a long needle syringe.
We do not recommend drying the cell before filling with your sample solution, but it should be rinsed twice with the buffer you are using for the experiment. Finally, it should be completely drained and filled with your sample solution in the manner described earlier. Because a small amount of the buffer used for final rinsing will adhere to the walls of the cell and act to dilute your sample solution, you may wish to correct for this by lowering your sample concentration by 2% if you measured concentration before the sample was introduced into the cell.

3.9 Cleaning Syringes.

Cleaning Injection Syringes
Disconnect the syringe from the pipette and insert the accessory plunger into the top of the syringe. Attach the plastic loading syringe tube to the filling port at the top of the syringe Insert the tip of the injection syringe into the cleaning solution and draw 2ml of solution through the syringe Empty the plastic loading syringe and repeat the above step at least two more times. Insert the tip of the injection syringe into distilled water and draw 2ml of solution through the syringe Empty the plastic loading syringe and repeat the above step at least two more times. Insert the tip of the injection syringe into HPLC Grade methanol and draw 2ml of solution through the syringe. Empty the plastic loading syringe and reattach it to the port. Remove the accessory plunger. Reattach the plastic loading syringe. Apply vacuum to the glass end of the injection syringe using the vacuum tube attachment for approximately 10 minutes to dry the methanol.

Conclusion

If the data you obtained using 2.5% methanol looks considerably worse than the sample data we have provided for comparison, then you should go back to the beginning of this tutorial and repeat it. Perhaps you might want to read the Troubleshooting section first. If you are satisfied with your methanol dilution data, then you may want to move on to real binding experiments.
4.3 Rnase-2CMP Experiment Tutorial
For the initial ITC tutorial runs using a biological sample, one trial test kit will be provided by MicroCal at no charge that contains buffer, the ligand 2'CMP and the enzyme Ribonuclease A. To order your trial test kit, please contact info@microcalorimetry.com with shipping information and it will be sent to you. Before your instrument was shipped from MicroCal, aliquots of the same samples were used to generate a binding isotherm and a copy of the results that were obtained will be included. The results will include the run parameters and the fitting parameters obtained from the data 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 instrument. The run parameters used in this experiment will be obtained from the rnase.inj run parameters file. This experiment should follow all recommended system preparation, sample preparation, loading and related operational standards outlined previously in this manual. You should expect your results to be close to the data set sent with the samples. The following percentage error data would be considered acceptable for a new user.

N (Stoichiometry)

Kb (binding Constant)

H (Enthalpy Change)

If everything has gone well to this point, then you should be ready to begin studies on your own samples. Good luck!!
Section 5: Instrument Maintenance
The maintenance requirements of the VP-ITC are simple, but essential for quality experiments. The cells, injection and filling syringes must be kept clean. The Plunger Tips for the Pipette Injection Syringe must be replaced regularly to prevent excess wear that would allow the titrant to leak from the syringe. Y-axis calibration is usually not a problem, but it 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 the bottom of the cell) should be approximately 3 mm. Once the syringe height adjustment is set, it is not usually a problem, but this distance is custom for each instrument. This means that the user must set the height position of newly purchased replacement syringes.

Setting the New ITC Syringe Height
7. Loosen the set-screw on the upper Syringe Clamp so that the disk is free to slide up and down on the outer diameter of the syringe glass. 8. Position the syringe in the syringe holder so that the tip of the syringe will not hit the bottom of the cell when the syringe/holder assembly is inserted into the sample cell. This can be easily accomplished by positioning the glass of the syringe so that it is flush to the bottom of the holder, but not lower. 9. Insert the syringe/holder assembly into the sample cell compartment and properly seat the assembly into place. 10. Gently push down on the top of the syringe until you feel it hit the bottom of the cell. If you are unsure then double-check it by lifting the syringe up and lowering it again until the bottom of the cell stops it.
11. Lower the upper Syringe Clamp so that it is flush to the top of the syringe holder. 12. Tighten the set-screw of the upper Syringe Clamp so that the disk is fixed to the syringe glass. 13. Raise the syringe by carefully prying up on the upper Syringe Clamp until it is approximately 3 mm higher than the top 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 so that it is again flush to the top of the syringe holder. 15. Tighten the set-screw of the upper Syringe Clamp and your syringe height is now fixed at 3 mm from the bottom of the sample cell.

Reassemble the Pipette

Please refer to Section 1.6 Pipette Assembly (page 9), for instructions on how to reassemble the pipette with the new syringe
5.5 Temperature Calibration
ITC Temperature Calibration Kit Contents
Stainless Steel capillary temperature standards (Qty. 2). Origin 5.0 project, ITCCalibration.opj, used for convenient data interpretation and for generation of calibration reports. VPITC Temperature Calibration Instructions.
ITC Temperature Calibration Procedure
You have received temperature standards for use with your VP-ITC. This document will describe how a VP-ITC user can use the capillary temperature standards to periodically check the calibration of the temperature data channel of the VP-ITC. It is recommended that the ITC temperature calibration be checked approximately once every 12 months. The measured transition midpoints should be within +/- 0.2 degrees of the known transition midpoints (28.2, 75.9 C), or adjustments to the temperature calibration coefficients should me made. Each of the 2 capillary temperature standards contains a wax with a known melting point. The shorter standard contains Octadecane (C18H38), which has a known melting point of 28.2 C. The longer standard contains n-Hexatriacontane (C36H74), which has a known melting point of 75.9 C. By heating these standards through their respective melting points while measuring the heat effect as a function of cell temperature, an ITC user can verify the proper calibration of the temperature measurement system of the VP-ITC. The procedure for performing a temperature calibration run, for analyzing the results, and for adjusting the calibration coefficients (when necessary) are described below:

6.2 Troubleshooting

PROBLEM: Long equilibration period before an experiment starts. There is no capability for directly cooling the cells nor the inner jacket of the VP-ITC, as was also the case for our earlier ITC instruments. If the operator is carrying out a series of experiments in the VP-ITC at 25.0 C, for example, and if the freshly loaded sample solution has a temperature of 26.0 C, then there are but two choices. If the No Check T option in the set-up window is checked X and the experiment started, then the system will equilibrate quickly at 26.0 C. If the No Check T option is unchecked then the software begins a process to cool the cells. This process consists of first lowering the temperature of the outer shield (which has cooling capability) far below 25 C which in time acts to lower the temperature of the inner jacket and ultimately the cells, both of which must cool by heat transfer to the outer shield. After the cells and inner jacket have cooled to 25 C, the temperature of the outer shield is cycled back up to its set position to begin the experiment. Because heat exchange with the outer shield is slow, this process can take a significant amount of time, even when the cell temperature only needs to be decreased by a degree or so.
AVOIDING THE PROBLEM: To avoid the lengthy delay in cooling 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, is at or below the temperature where the experiment will be carried out. When this is the case, the No Check T option should be left unchecked and the cells will promptly be heated to the correct temperature (without changing the temperature of the outer shield) so that the experiment will be ready to begin in a short time. For experiments below room temperature, solutions should initially be cooled (after degassing) significantly below the set temperature since they will warm up somewhat when drawn into the loading syringe and transferred into the cell.
PROBLEM: Baseline is stable without stirring, but becomes unstable after syringe is inserted and stirring begins prior to injections. SOLUTION: Indicates a stirring problem. May be debris or bubbles in cell, bent injection syringe needle, improper syringe height adjustment, or injection system alignment problem. Debris or bubbles refill cell after rinsing or using cell cleaning apparatus and try again. Extraordinary circumstances may require carefully inverting the entire VP-ITC unit on a suitable stand and flushing the sample cell with the filling syringe to remove solid debris. If your test samples are suspensions of particles try higher stirring rate to achieve uniform suspension, although the extra noise caused by the increased stirring speed may cause deterioration of the baseline.