MicroCal Inc. Micro Calorimetry System

MicroCal Inc. Micro Calorimetry System User's Manual The following manual describes the hardware and software components of your new MCS system, including system setup, operation of the system, as well as appropriate techniques necessary to maximize the performance of your instrument. The manual is intended for both DSC (Differential Scanning Calorimeter) and ITC (Isothermal Titration Calorimeter) customers. The majority of this manual is relevant to all MCS users and should be read prior to operating the system. DSC customers may ignore Sections 5, 7.1, as well as Appendices A and B, while ITC customers may ignore Sections 6, 7.2 as well as Appendices C and D. All customers may access technical support from MicroCal by calling the toll-free MCS Technical Support Hotline, at 1-800-633-3115. Additionally, MicroCal encourages feedback from all our users so that we may continue to provide you with the highest quality Micro Calorimeters, while maintaining simplicity for the user!

Table of Contents

Section 1: Introduction to MCS System Components.......................................................................... 1 1.1 MCS System......................................................................................................................... 2 1.2 Differential Scanning Calorimetric Unit........................................................................... 3 1.3 Isothermal Titration Calorimetric Unit ............................................................................ 5 1.4 External Water Bath........................................................................................................... 7 Section 2: Setting Up Your MCS System .............................................................................................. 9 2.1 MCS Software Setup........................................................................................................... 10 2.2 MCS Hardware Setup......................................................................................................... 14 Section 3: Introduction to MCS Software ............................................................................................. 18 3.1 What is MCS Observer....................................................................................................... 19 3.2 Getting Started with MCS Observer ................................................................................. 21 Section 4: Using MCS Observer............................................................................................................. 23 4.1 Observer Data Display........................................................................................................ 24 4.2 Observer Menu Options ..................................................................................................... 26 Section 5: Running an ITC Experiment ................................................................................................ 34 5.1 Using Observer -ITC........................................................................................................... 35 5.2 Designing ITC Experiments ............................................................................................... 41 5.3 Sample Preparation ............................................................................................................ 45 5.4 Cell Loading......................................................................................................................... 46 5.5 Injection Syringe Filling ..................................................................................................... 47 5.6 Cell and Syringe Cleaning .................................................................................................. 49 5.7 Below Room Temperature Operation ............................................................................... 51 5.8 Far Above Room Temperature Operation ....................................................................... 52 5.9 Precautions........................................................................................................................... 53 5.10 Troubleshooting................................................................................................................. 54 5.11 ITC Experimental Tutorial .............................................................................................. 56 5.12 Maximizing Baseline Stability.......................................................................................... 75 5.13 Selecting the Proper Stirring Rate................................................................................... 76 Section 6: Running a DSC Experiment ................................................................................................. 77 6.1 Using Observer-DSC........................................................................................................... 78 6.2 Sample Preparation ............................................................................................................ 83 6.3 Cell Loading......................................................................................................................... 84 6.4 Cell Cleaning........................................................................................................................ 85 6.5 Sideways Operation for Precipitating Proteins ................................................................ 87 6.6 Troubleshooting................................................................................................................... 88 6.7 DSC Experimental Tutorial ............................................................................................... 89 6.8 Maximizing Baseline Repeatability ................................................................................... 104 Section 7: Calibrating the Cells.............................................................................................................. 105 7.1 ITC Cell Calibration ........................................................................................................... 106 7.2 DSC Cell Calibration .......................................................................................................... 108 Section 8: MCS Remote Control Customer Support ........................................................................... 110 Appendix A: ITC Cell Status Definitions .............................................................................................. 111 Appendix B: ITC Calibration Constants............................................................................................... 113 Appendix C: DSC Cell Status Definitions ............................................................................................. 116 Appendix D: DSC Calibration Constants ............................................................................................. 118

Section 1-Introduction to MCS

Section 1

Introduction to MCS System Components Section Contents: 1.1 MCS System 1.2 DSC Calorimetric Unit 1.3 ITC Calorimetric Unit 1.4 External Water Bath

-1-

Section 1.1-MCS System

Section 1.1

MCS System The MCS consists of a Controller Unit, one or two Calorimetric Units, one or two refrigerated water baths, a host personal computer, and associated accessories. The Calorimetric Units may be either Isothermal Titration Calorimetric (ITC) units or Differential Scanning Calorimetric (DSC) units. Any combination of one or two Calorimetric Unit types is allowed. The combination of types is specified via jumpers within the control unit. The host computer may be any system that is capable of running Microsoft Windows, and must have at least one available serial port (although two is strongly recommended if a serial mouse is to be used). The Controller Unit communicates with the host PC with an RS-232 serial link. All Calorimetric Unit operation is supervised directly by the Controller Unit. The host PC serves as user interface to the Controller Unit sending the command sequences needed to initiate experiments, and for displaying all generated data.

-2-

Section 1.2-DSC Unit

Section 1.2

Differential Scanning Calorimetric Unit The DSC Unit directly measures heat capacity of small liquid samples as a function of temperature. The normal operating temperature range of the unit is 0 deg. C. to 120 deg. C. however specialized units are available to extend the range from -60 deg. C. to 140 deg. C.. Scan rate is user specified from 0-120 deg/hr. for upscans and 0-45 deg/hr for downscans (limited by the bath units cooling capacity). All wetted surfaces are tantalum or teflon. Sample and reference cells are accessible for filling and cleaning beneath a twist off cap on the top of the unit. The sample cell is on the right as one faces the front of the unit. A teflon spill guard surrounds the cap and provides a storage area for beakers. The refrigerated water bath hose connections are made at the rear of the unit. Valves internal to the DSC unit control whether or not liquid circulates. The bath is used to cool the unit rapidly after heating and also for downscanning. Two 12v sealed lead-acid batteries are accessible through a removable plate on the bottom of the unit. The batteries should only require replacement every 3-4 years.

A pair of identical coin shaped cells is enclosed in an adiabatic 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 1.4 ml per cell. One cell serves as the sample cell and is normally filled with a dilute biological substance (e.g. 1 % protein and 99 % water). The other cell serves as reference and is normally filled with an identical volume of solvent (e.g. 100 % water). Both cells' outer surfaces are coated with resistive heating elements. A pressure of 20 psi is applied to the samples during scans to prevent sample boiling. The two cells are heated with a constant power input to their main heaters during a scan. The reference cell receives slightly more power through a second heater, the reference offset, than the sample cell. The temperature difference between the two cells is constantly measured and the cell feedback system proportionally increases or decreases the sample cell's power

-3-

Section 1.2-DSC Unit input through it's feedback heater in an effort to keep the temperature difference very close to zero. Since the masses and volumes of the two cells are matched the power added or subtracted by the cell feedback system is then directly a measure of the difference between the heat capacity of the sample and reference solutions. A signal proportional to that cell feedback power is called CFB, and with the instrument temperature and time, constitutes the instrument's relevant raw data. The CFB is calibrated in units of mCal/min. The operating range is 0 to 140 mCal/min. If the CFB is negative there is no feedback power applied to the sample cell and no measurements of heat capacity are being made. An adiabatic jacket scans along in temperature with the two cells providing the required thermal environment for ultra sensitive measurements. A jacket feedback system constantly monitors the temperature difference between the cells and the jacket. Power applied to the jacket resistive heater is proportionally increased or reduced to keep the temperature difference small. The temperature difference is presented as secondary data and called ∆T. The jacket feedback current, JFBI, is available as secondary data. The feedback power for the sample cell and jacket normally constitutes a small fraction of the total power dissipated in each. The set of main heaters located on both cells and the jacket dissipate relatively large constant powers so that sensitive measurements may be made at high scan rates. The cell main heaters receive the cell main current, CMI. The jacket main heater receives the jacket main current, JMI. CMI and JMI are measured throughout experiments and are available as secondary data. A set of scan rate parameters are used by MCS Observer software to compute the heater settings for CMI, JMI, and the reference offset power from user specified scan rates. In practice a differential scanning micro calorimeter is inevitably off balance slightly due to imperfect matching of the sample and reference cells. The result is that there always exists a reference baseline for a given experimental scan which is subsequently subtracted from the experimental data to yield ∆Cp for a given sample. The instrument is calibrated by changing the power input to the reference cell by a known amount and mapping the resulting deflection in the cell feedback power signal to the known power via a scale factor. Downscans are done by linearly decreasing the temperature of the refrigerated bath which circulates through the walls of the adiabatic jacket. The cells cool by conducting heat to the jacket. Because of the inefficiency of the conduction the cells normally trail the jacket in temperature by several degrees, depending on downscan rate. Since instrument temperature is measured at the jacket the ∆T is also relevant raw data during a downscan so that the two temperatures may be added to give sample temperature. The cell feedback system operates as during an upscan. The CFB power and the reference offset power are the only heat applied during a downscan. The cell and jacket main heaters are turned off and the jacket feedback system is not used.

-4-

Section 1.3-ITC Unit

Section 1.3

Isothermal Titration Calorimetric Unit The ITC Unit directly measures heat evolved or absorbed in liquid samples normally as a result of injecting precise amounts of reactants. A spinning syringe is utilized for injecting and subsequent mixing. Spin rates are user selectable. The normal operating range is 0 deg/C to 80 deg/C. Wetted surfaces are Hastelloy-C alloy. Detailed corrosion data for Hastelloy-C is included in the MCS manual. The general rule is that no strong acids should be put in a ITC unit. 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. Two 12v sealed lead-acid batteries are accessible through a removable plate on the bottom of the unit. The batteries should only require replacement every 3-4 years.

A pair of identical coin shaped cells is enclosed in an adiabatic 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 1.7 mL. per cell. During an experiment the reference cell is heated by a very small constant power, the reference offset. The temperature difference between the two cells is constantly measured and a proportional power is increased or reduced to the sample cell by the cell feedback system to keep the temperature difference very small. A signal proportional to that cell feedback power is called CFB, and with the instrument temperature and time, constitutes the instrument's relevant raw data. The CFB is calibrated in units of µCal/sec. The operating range is 0 to 100 µCal/sec. The instrument is calibrated by changing the power input to the reference cell by a known amount and mapping the resulting deflection in the cell feedback power signal to the known power via a scale factor. An injection which results in the chemical evolution of heat within the sample cell causes a peak downward in the cell feedback power since the heat evolved chemically provides heat that the cell feedback is no longer required to. The opposite is

-5-

Section 1.3-ITC Unit true for endothermic reactions. Since the cell feedback has units of power, the time integral of the peak yields a measurement of thermal energy, ∆H. If the CFB is negative there is no feedback power applied to the sample cell and no measurements of thermal energy are being made. If the CFB saturates positive data will appear clipped. Either can happen during an experiment if a sample generates too large a signal. It is then useful to be able to offset the CFB power level within it's operating range. This is done by adjusting the reference power setting called Exothermic Headroom prior to the repeat experiment. A jacket feedback system constantly monitors the temperature difference between the cells and the jacket. Power applied to the jacket resistive heater is increased or reduced to keep the temperature difference small. The temperature difference is presented as secondary data and called ∆T. The jacket feedback current, JFBI, is available as secondary data. This scheme can only function when the instrument is above ambient temperature since temperatures are controlled only by applying heat in various amounts. In order to work below room temperature, water-cooled plates surround the core instrument, and permit simulation of lower effective ambient temperatures. A refrigerated circulating bath provides fluid to the plates. Hose connections are made at the rear of the unit. An independent set of cell main heaters is used for thermostatting purposes between experiments and also for increasing instrument experimental temperature. The cell main current, CMI, is available as secondary data.

-6-

Section 1.4-External Water Bath

Section 1.4

External Water Bath This section is intended to familiarize the user with the Haake external water bath's controls and connectors. This is not intended to, nor does it suffice as a replacement for the user's manual which you have received with the Haake. Please refer to the following diagrams for a minimum description of the Haake F3-CH connections and controls:

Identification of Parts-Front Side:

(1) (2) (3) (4) (5) (6) (7) (8)

Main Switch with Power Control Light Heater Control Light Digital Switch for Preset Temperature Setting Reversing Switch (Internal/External) Digital Display for Current Temperature of Circulating Coolant Malfunction Indicator Release Switch Excess Temperature Limiter

Considerations & Precautions: 1.) Fill the bath with a 50% water, 50% antifreeze mixture. 2.) Care should be taken so that no solids or particulates are allowed into the circulation liquid. 3.) Do not turn on the water bath until the MCS Control Unit is powered up. 4.) Turn off the bath power whenever the MCS Control Unit's power is off. 5.) Always have the Reversing switch in the External position (pushed in) when using the bath with the MCS system. This allows the MCS Control Unit to control both the set temperature, and the circulation of the bath. 6.) Any other controls and/or switches present on the Haake will be preset by MicroCal, and needn't be adjusted.

-7-

Section 1.4-External Water Bath

Identification of Parts-Rear Side:

(1) (2) (3) (4) (5) (6) (7) (8)

Socket for connecting external temperature controller Socket for connecting control cable to a refrigerated bath vessel Socket for monitoring functions of safety elements Coolant Out Port Coolant In Port Fuses Main Power Cable Selector Switch - Control Mode (INT/EXT)

Considerations & Precautions: 1.) All of the Haake sockets which receive cables are unique! You can be assured that you are cabling correctly if the socket accepts the cable in question (without force!). A quick visual inspection of both the cable end and the socket should help you to find the correct socket for each cable. 2.) The bath cable which came with your MCS system (gray cable with one connector on one end and two on the other) connects to sockets (1) and (3), as well to the MCS Control Unit socket labeled 'Cell # Bath' at the other end. 3.) The @ 2 ft. gray cable which came with your Haake connects on one end to socket (2), and to the refrigerated vessel socket at the other end (rear of refrigerated vessel). 4.) Plug the main power cable into the AC power receptacle (located at the rear of the refrigerated vessel), and then plug the AC power cable which exits the rear of the refrigerated vessel into it's own AC source (separate from the power for the Controller, Calorimetric Units, Host Computer,...). 5.) Use the black tubing and insulation provided to plumb your Haake to the appropriate calorimetric units you have purchased. The Coolant Out port at the Haake gets connected to the Bath In port at the calorimetric unit. The Coolant In port at the Haake gets connected to the Bath Out port at the calorimetric unit. Be sure and use the hose clamps provided when making these connections. 6.) The Selector Switch must remain in the preset Internal position.

-8-

Section 2-MCS Setup

Section 2

Setting Up Your MCS System Section Contents: 2.1 MCS Software Setup 2.2 MCS Hardware Setup

-9-

Section 2.1-MCS Software Setup

Section 2.1

MCS Software Setup Installation If you purchased your host computer from MicroCal, all necessary software will already be installed. If not, you should install your software by running the software installation program that came with your MCS distribution disks. The installation program copies all of the necessary files onto your hard disk and you should now be ready to run the software. The following is a description of the files that were copied to your hard disk during the installation process. WARNING Altering any of the files provided with the MCS system may cause the system to be inoperable. Any changes that the user may make, should take place from the MCS Observer, as explained in Section 3. Directory C:\MCS\ OBSERVER.EXE The user interface program. This is the program that allows the user to operate the MCS system. Experiments can be executed, data collected from the MCS Control Unit and displayed in Origin, and MCS peripherals (water bath, desiccator,...) may be controlled through the MCS Observer. Please refer to the Using Observer topic of help for a thorough explanation of the Observer.EXE program. *.VBX, *.DLL Files with either of these extensions are Windows DLL's (Dynamic Link Libraries). These files contains procedures/functions that are called by the MCS Observer. OBSERVER.HLP The compiled help file for accessing help from the MCS Observer. Directory C:\MCS\SUPPORT\ Z.BAT A DOS batch file that is called during the download process. This file will only be executed if the MCS system files have been changed since the last time this batch file was executed. PKZIP.EXE Program used to compress files. All files that get downloaded to the MCS Control Unit are compressed into one file by this program and then transferred. This program is called in the Z.BAT (see Above) program. This is a general file compression program distributed by PKWARE, Inc.. LASTRPB1.RUN Whenever an experiment (run) is begun at Cell #1 of the MCS all of the run parameters for that particular run are saved to this file. This file also maintains a log of runs completed, so that any unretrieved data may eventually

-10-

Section 2.1-MCS Software Setup be retrieved. When the MCS Observer is run it reads this file in order to restore the run parameters to their previous values, and to determine if there is unretrieved data at the MCS Control Unit. LASTRPB2.RUN Identical to LASTRPB1.RUN, but for Cell #2. See Above. DATATX.CON This file contains communications parameters used by Observer.exe. The contents of this file should not be of concern to the users, and this file should not be edited or deleted. COM.NUM This file contains the communications port number which will be used for all MCS serial communications. Directory C:\MCS\SETUP\ RNASE.INJ (Only present for ITC customers) The ITC experimental setup file used to generate the RNASE.ITC datafile (see directory C:\MCS\DATA below). ITCCALIB.CAL (Only present for ITC customers) The ITC calibration experimental setup file used to generate the ITCCALIB.ITK datafile (see directory C:\MCS\DATA below). REF.SCN (Only present for DSC customers) The DSC experimental setup file used to generate the REF30.DSC, REF60.DSC and REF90.DSC datafiles (see directory C:\MCS\DATA below). DSCCALIB.SCN (Only present for DSC customers) The DSC experimental setup file used to generate the DSCCALIB.DSC datafile (see directory C:\MCS\DATA below).

Directory C:\MCS\DATA\ *.ITC ITC data files.

-11-

Section 2.1-MCS Software Setup *.ITK ITC Calibration data files. *.ITC DSC data files. The following datafiles were generated with your instrument, at MicroCal. The contents of these files are described below. RNASE.ITC (Only present for ITC customers) Datafile generated with your ITC Cell unit, at MicroCal. Experiment consists of injecting cytidine 2'monophosphate (2'CMP) into ribonuclease A. A test kit with duplicate samples has been included with your system so that you can repeat this experiment and compare your results to this datafile. The RNASE.INJ setup file was used to generate this datafile. ITC users are encouraged to use the RNASE.INJ setup file when duplicating the Rnase/2'CMP experiment. ITCCALIB.ITK (Only present for ITC customers) ITC calibration datafile generated with your ITC cell unit, at MicroCal. REF30.DSC (Only present for DSC customers) A water-water scan at 30 deg/hr generated with your DSC cell unit, at MicroCal. REF60.DSC (Only present for DSC customers) A water-water scan at 60 deg/hr generated with your DSC cell unit, at MicroCal. REF90.DSC (Only present for DSC customers) A water-water scan at 90 deg/hr generated with your DSC cell unit, at MicroCal. DSCCALIB.DSC (Only present for DSC customers) DSC calibration datafile generated with your DSC cell unit, at MicroCal. Directory C:\MCS\MCSZIP\ MONITOR.EXE CELL1CAL.CON CELL2CAL.CON *.CMD Core elements of MCS OS. Directory C:\MCS\HARDWARE\ Logs of instrument hardware histories. The files that are provided for Origin DDE are the following:

-12-

Section 2.1-MCS Software Setup Directory C:\Origin\ MCSDATA.ORG This is the Origin document that will be used for viewing MCS data. Users should not alter any of the worksheet or plot window names contained in this document. DSCPRERN.OTP This is an Origin Plot Window Template. Specifically, this template is used to display all prerun data that may be generated by an MCS DSC Unit. DSCRUN.OTP This is an Origin Plot Window Template. Specifically, this template is used to display all run data (filtered experimental data) that may be generated by an MCS DSC Unit. ITCPRERN.OTP This is an Origin Plot Window Template. Specifically, this template is used to display all prerun data that may be generated by an MCS ITC Unit. ITCRUN.OTP This is an Origin Plot Window Template. Specifically, this template is used to display all run data (filtered experimental data) that may be generated by an MCS ITC Unit. MCS.CNF This file contains all of the Macros (Labtalk Scripts) that are needed for using Origin with the MCS system. Users who feel confident using Origin templates should feel free to edit any of the graphical characteristics of these .OTP templates (color, text size, fonts, ...), as described in the Origin Tutorials for DSC and ITC.

-13-

Section 2.2-MCS Hardware Setup

Section 2.2

MCS Hardware Setup The MCS requires table or bench top arrangement so that the Control Unit, the Calorimetric Unit(s), and the refrigerated water bath(s) are all on the same level. The Control Unit should be situated with a Calorimetric Unit on either or both sides of it and the baths on the other side of the Calorimetric Units.

It is desirable, if possible, that the water baths are placed on the same level as the Calorimetric units since the circulation pumps circulate the water more quickly. This is especially important for DSC downscanning. Do not place an exhaust breeze from a refrigerated bath closer than 12 inches to a calorimetric unit. All of the Units should be handled carefully. Ventilation space of at least 12 inches should be provided behind the Control Unit and the baths. The front ventilation intakes of the Control Unit must be free to draw air. The environment should be well thermostatted for highest instrument performance. Large room temperature variations will effect the repeatability of the DSC Unit reference baseline and will cause the ITC Unit reference baseline to drift. Both Calorimetric Unit types rely on internal voltage measurements being made with resolutions on the order of a billionth of a volt. Battery operation and extensive shielding is employed to protect these measurements from external sources of interference. However some care at initial location choice is required. The two main concerns are radiated AC magnetic fields and equipment which corrupts the quality of the AC power source for the MCS. The best way to provide AC power to the MCS is by plugging the MCS units and water baths into a single power strip. If AC power surges and or sags are a problem standard computer system uninteruptible power supplies or line conditioners may be used to provide the power to the strip. Make all cable connections before plugging the units into the AC power mains. After your units are in place as described above, locate all of the cables that came with your instrument. You will find the following cables, all of which are clearly marked. CABLE/PARTS LIST: - Main cell cables, 1 for each calorimetric unit included in your system. - Bath cable, 1 for each calorimetric unit included in your system. - RS-232 cable with phone jack on both ends. - 9 and 25 pin connectors (accepts male phone jack) for connecting your RS-232 cable to a serial port at the rear of the host computer. - 1 power cord for the MCS Controller. - 1 power cord for each calorimetric unit included in your system. - 2 lengths of bath hose and hose insulation for each calorimetric unit included in your system. - 4 hose clamps for each calorimetric unit included in your system.

-14-

Section 2.2-MCS Hardware Setup - 1 'long' length of tubing and 2 hose clamps for each DSC calorimetric unit included in your system. This tubing is used for connecting a Nitrogen source to your DSC cell ports.

MCS Control Unit-Rear View

-15-

Section 2.2-MCS Hardware Setup

DSC Calorimetric Unit-Rear View

ITC Calorimetric Unit-Rear View

-16-

Section 2.2-MCS Hardware Setup Connect the Calorimetric Unit(s) to the Control Unit with the main cables which plug in at the rear of the units. Make sure the cables are inserted all the way into their connection locations. Connect the bath(s) to the Control Unit with the bath cables. Connect the bath(s) to the Calorimetric Unit(s) with the hose clamps provided. The insulation provided must be used and is purposely cut longer than the hoses to expand into place after installation. Do not alter the hose or insulation lengths without consulting MicroCal. Make sure the output hose line from the bath connects to the Calorimetric Unit input. Connect the Control Unit to the host PC with the RS-232 modular cable. Use the appropriate 9 or 25 pin adapter for the serial port of your host PC. Connect the AC power cords to each of the MCS Units. The Calorimetric Units have no power switches. They are constantly charging their batteries and the motors in the ITC unit are energized when plugged in Do not switch the Control Unit on until instructed to do so by the MCS software at the host PC. Connect the DSC pressure port on the top of the instrument to a nitrogen tank regulator. Do not turn the bath(s) on until the Control Unit has been turned on. The Haake bath units have a switch on the front panel which determines whether the bath is to be controlled internally by the temperature setting dials on the bath unit or externally by the MCS. Always set this switch to the external setting when using with the MCS.

-17-

Section 3-Introduction to Software

Section 3

Introduction to MCS Software Section Contents: 3.1 What is MCS Observer 3.2 Getting Started With MCS Observer

-18-

Section 3.1-What is MCS Observer

Section 3.1

What is MCS Observer MCS Observer is the software at the host computer which acts as the user interface to the MCS Control Unit. Observer is used to collect data from as well as to issue command sequences to, the MCS Control Unit. MCS Observer is capable of operating two calorimetric units (DSC or ITC) independently as well as simultaneously. When MCS Observer is opened it first establishes communications with the MCS Control Unit and asks the for the current number and types of cells (cell configuration) that are connected to the Control Unit. This information is coded into a series of jumpers located within the Control Unit. The MCS Control Unit's operating system is stored on disk at the host computer. It must be downloaded to the Control Unit each time the Control Unit is powered up. This is done by choosing the Download MCS OS option. Once the Control Unit has received the MCS OS it is capable of operating with or without the presence of the MCS Observer. Closing the MCS Observer or turning off the host computer has no effect on the Control Unit. Turning off AC power to the Control Unit will clear MCS OS from memory. The MCS Observer must be running if the user wants to see or save, in real time, the data being generated at the Control unit. It is important to understand that data which is generated at the Control Unit remains available to the user, and needn't be collected by the host as it is generated. This allows the host computer to be turned off, or for MCS Observer to be closed without losing any experimental data. MCS Observer is a multitasking application, and users should feel free to run other Window's applications simultaneously.

MCS Observer and Origin In addition to communicating with the MCS Control Unit, the MCS Observer will also be communicating with MicroCal's data plotting and analysis application called Origin. Data that is collected at the host is sent to Origin via DDE (Dynamic Data Exchange) and is plotted for user viewing. Text command sequences called Scripts are also sent to Origin via DDE for controlling the appearance of plot windows, and for various other tasks. When a command is sent to Origin by the MCS Observer a message (Executing Origin Scripts) appears in the Cell Status box for that particular cell. Though Origin need not be used, it is the only means of viewing plotted data in real time and is an integral part of the system's normal operation. Origin is also used for all data analysis on MCS data files. Because Origin is used for both data display and data analysis, there are a few things to keep in mind.

Origin For Real-Time Data Display DDE allows separate Windows' applications to exchange information. It is through DDE that the MCS Observer is able to make use of Origin's powerful graphing capabilities. All data generated at the MCS Control Unit can be displayed real time in Origin using DDE. Because Origin within Observer is dedicated to data collection and data display through DDE exchange, it is not generally available for doing other tasks contained within it's menus, such as calling up other Origin documents, saving documents, printing, analysis of data, etc. Attempting to do this can cause Origin to crash (even so, you would not lose your data nor would the your experiment be aborted) because of the inability to timeshare properly within the same application. If you need to do these other tasks while live data is being generated in Origin within Observer, then you should open another version of Origin from Program Manager in the usual way and there will be no problem. You should also be aware that the MCSDATA.ORG document should be dedicated to Origin within Observer. This should not be called into a second Origin application while data is being collected. This document also should never be modified in any way lest it be made inappropriate for the Observer application.

Origin For Data Analysis

-19-

Section 3.1-What is MCS Observer When used for data analysis in a separate window, the user is unrestricted in what he/she can ask Origin to do. The only restriction in this case is that the user should not use the MCSDATA document as that document should be dedicated to DDE. Please refer to the Origin manual for a full description of it's use for data analysis.

-20-

Section 3.2-Getting Started With MCS Observer

Section 3.2

Getting Started with MCS Observer Once the 'MCS OS' files are received by the MCS Control Unit two things will happen. First the MCS OS will start operating the MCS Control Unit. This is verified by noting the periodic blink of the µP light on the front of the Unit. Second, the MCS Observer will go through some initialization procedures.. MCS Observer first attempts to establish communications with the MCS Control Unit and then obtains the number and types of Units (Cell Configuration) that are connected to the MCS Control Unit. The main window of Observer opens allowing the user access to the instrument control menus and displays live instrument data for the cell units which are connected.

Problems Getting Started Problem: The MCS Observer status indicator never indicated that a file transfer occurred. The % complete indicator never increased or never appeared after choosing the 'Download' option.. Solution1: Check to make sure that your MCS Control Unit is cabled to a valid Com Port at the back of your computer. If it is, make sure that the Com Port that was opened is the same as the Com Port that you are cabled to. Solution2: Be sure that the Control Unit power is not turned on until prompted for. Solution3: Check to make sure that the cable from the host computer's Com Port is cabled to the phone jack labeled 'Host' at the rear of the Control Unit. Problem: The MCS Observer responded with 'No MCS Unit Found', but all other indications are that the file transfer was successful, and that the MCS OS is running (flashing µP light). Solution: Try running the MCS Observer again (or choose 'Try Again' when prompted ) and see if you get the same result. Problem: The MCS Observer never indicated that a Com Port was successfully opened. Solution1: If you changed the Com Port that you are using for MCS communications since you installed the software, then you will need to edit a file. From Notepad or any other text editor, open the file C:\MCS\SUPPORT\COM.NUM. You should see one entry in this file, and it is the Com Port number that will be used for all MCS communications. Edit the ComPort entry to agree with the one you would like to be using, and are cabled to. Save the changes and try again. Solution2: Try exiting and then restarting Windows. Now try Observer again.

-21-

Section 3.2-Getting Started With MCS Observer

Command Line Options When launching a program from Windows' Program Manager you can specify what's called a command line option. The command line option is additional text placed after the program name, which is then passed to and interpreted by the program that is run. The following command line options are available when running the MCS Observer, and all are case insensitive: +O, -O, /O, or \O: Any of these will cause Origin to be opened along with the MCS Observer. Once opened, a DDE link will automatically be established and all prerun and run data will be plotted in Origin for user viewing. +R1, +R2: The +R command line tells MCS Observer that all unretrieved data at the Control Unit should be downloaded to the host when MCS Observer is launched. The number that follows the +R corresponds to a cell number in your MCS system. Thus, +R1 tells Observer to retrieve all data at Cell #1, without prompting the user to do so. -R1, -R2: The -R command line tells MCS Observer that all unretrieved data at the Control Unit should not be downloaded to the host when MCS Observer is launched. The number that follows the +R corresponds to a cell number in your MCS system. Thus, -R1 tells Observer not to retrieve all data at Cell #1, without prompting the user to do so. This option is primarily for use by MicroCal, and is not recommended. The installation program will set up the following command line for your MCS Observer icon in Program Manager: Observer +O +R1 +R2.

-22-

Section 4-Using MCS Observer

Section 4

Using MCS Observer Section Contents: 4.1 Observer Data Display & Definitions 4.2 Observer Menu Options

-23-

Section 4.1-Observer Data Display

Section 4.1

Observer Data Display This window normally displays only the default data channels (Time, CFB, ∆T, BathT, Cell Status) but user may also select to display the heater current data as well (JFBI, JMI, CMI). Generally, the heater current data is of no interest to the user and is available only to confirm proper function of heater systems. See picture below along with definitions for a full explanation of all the MCS data channels.

Time Displays the current time (seconds) into a prerun or a run. Time data will be reset to zero whenever equilibration for a new run is started , and again after equilibration is complete and the actual run has begun. When a ITC/DSC Unit is in it's idle state ('Cell Idle' for DSC, 'Thermostatting' for ITC) or any postrun state, the time data will always be zero.

Temperature (Temp) Displays the current jacket temperature.

Cell Feedback (CFB) Displays data which is proportional to the feedback power to the sample cell.

Delta T Displays the temperature difference between the jacket and the two cells (sample and reference). Positive ∆T means the jacket is cooler than the cells.

-24-

Section 4.1-Observer Data Display

Bath Temperature (BathT) Displays the current temperature (deg. C) of the external water bath. This is the current bath temperature, not the bath's target temperature. The current target temperature may be accessed and changed for a particular cell as described later.

Cell Status Displays the current state of the cell. Every experiment consists of a sequence of instrument states unique to DSC and ITC. The descriptions of the different states are found in the appendices.

Optional Displayed Channels Jacket Feedback Current (JFBI) The amount of current (amps) passing through the JFB heater. Positive values indicate that the heater is on, while non-positive values indicate the heater is off.

Jacket Main Current (JMI) The amount of current (amps) passing through the jacket main heater. Positive values indicate that heater is on, while non-positive values indicate the heater is off.

Cell Main Current (CMI) The amount of current (milliamps) passing through the cell main heater. Positive values indicate that heater is on, while non-positive values indicate the heater is off.

-25-

Section 4.2-Observer Menu Options

Section 4.2

Observer Menu Options Options Opens the MCS Options drop down menu.

Allows user access to several menu options. Options include the appearance of the main window, whether or not data is to be displayed in Origin, as well as access to various other system peripherals.

Cell Display Allows user to display Cell 1 data, Cell 2 data or data from both cells. When only one cell is currently displayed, users may toggle directly to the other cell's display by double clicking on the cell status box (the bottom most text box) of the currently displayed cell.

Data Channel Display Allows user to display only the default data channels (Time, CFB, ∆T, BathT, Cell Status) or to also display the heater current data as well (JFBI, JMI, CMI). Generally, the heater current data is of no interest to the user and is available only to confirm proper function of heater systems. For a full description of the individual data channels, see the MCS Software:Data Channel Definitions section of help.

Data Display in Origin Allows user to turn off/on the DDE (Dynamic Data Exchange) link for a particular cell. When this menu option is checked then DDE for that cell is on and all prerun and run data will be plotted in Origin for user viewing. When Origin is opened from Observer (command line option, or menu option) the Data Display is turned on for each cell that is connected to the MCS control unit. The DDE link remains on until the user turns it off via the menu

-26-

Section 4.2-Observer Menu Options or closes Origin. Once turned off, turning the DDE back on will cause any currently displayed data to be cleared from the Origin worksheet and Data Display will pick up from the next received data point. Depending on the type of cell and the current cell status, the data that is displayed in Origin will differ as follows: Cell Type: Cell Status: Displayed Data: X Axis: ITC,DSC Thermostatting, Cell Idle None N/A ITC Checking Baseline CFB ITC All Other Prerun States CFB,JFB,Temp DSC All Prerun States CFB,JFB,Temp,BathT ITC Collecting Injection # CFB DSC Scanning-Scan # CFB ITC,DSC All Postrun States None N/A

Time Time Time Time Temp

Open Origin Normally, Origin will be opened along with Observer via the Program Manager. By including a "-O" or "/O" or "\O" or "+O" (case insensitive) in the command line, Origin will automatically be opened with Observer and the DDE links will be established for all cells connected to the MCS unit. This is the easiest way to use Origin for data display, and is recommended. However if you do open Observer without opening Origin then this menu option, Open Origin, may be used and DDE links will be established for all cells connected to the MCS Control Unit. If Origin is opened with Observer but then Origin crashes or is closed for some reason, the Open Origin option will not be available, and cannot be used to reopen Origin. In such a case, the easiest way to recover from the crash is to go to the Program Manager (if Origin has crashed, it is a good idea to exit Windows and then restart windows) and double-click on the MCS Observer icon. Your experiment will have continued to run on schedule as if the crash had not occurred, and no data is lost. IMPORTANT: It is frequently convenient to open and use a second copy of Origin for data analysis, while Observer is running and DDEing data to the first copy of Origin. There is no problem in doing this as long as the second copy of Origin is not used to open the default DDE document (MCSDATA.ORG). Since the first copy of Origin is relatively busy processing DDE data and commands it should not be used for any data analysis but should be left dedicated to DDE data display.

Constants Calibration Constants Displays all the calibration constants for all cells connected to the MCS control unit. The calibration constants that will appear will be determined by the types of cells that are present (DSC or ITC).

-27-

Section 4.2-Observer Menu Options Advanced DSC Constants Displays infrequently changed DSC system constants for each DSC cell included in your MCS system. If there are no DSC Calorimetric Units included in your system then this menu option will not be available.

Exit Exits the MCS Observer program. Data being generated at the MCS will be buffered and retrieved the next time Observer is opened. Data stored at the MCS will remain there until the Control Unit is turned off.

ITC Cell (Cell 1 or Cell 2) Opens the ITC drop down menu.

Contains all the main menu options for an ITC cell. Provides access to all windows necessary to calibrate the cell, setup and carry out an ITC experiment. Also accesses control of ITC cell peripherals (water bath, desiccator).

ITC Setup Opens the ITC Setup Window. Refer to Section 5.1 for a complete description.

-28-


Calibration Setup Opens the ITC Calibration Setup Window.

Pictured above is the ITC Calibration setup window. It is here that users will begin all ITC Calibration experiments. User defines the run parameters and transmits them to the Control Unit. The setup window is also used to create/edit/save run parameter files (setup files). Once all parameters are as desired, the file can be saved with the File:Save menu option. Once saved, the same run parameters can be loaded (at some later date) into the Setup window with the File:Load menu option. Data files may also be used in the same way in order to quickly set up a new experiment using the same set parameters used for the old data file. When loading a data file into the setup form the run parameters are contained in and extracted from the data file header.

Run Parameters Defined: Number of Pulses Total number of pulses for a given ITC calibration experiment. Exper. Temp (Experimental Temperature) User inputs the desired experimental temperature. Initial Delay Time (sec.) into experiment before beginning the first pulse. Pulse Rate The desired calibration pulse rate (µcal/sec). Pulse Duration The duration (sec.) of the calibration pulse. The pulse rate multiplied by the pulse duration(µcal/sec) yields the total energy (µcal) added to the ITC Cell's reference cell.

-29-

Section 4.2-Observer Menu Options Time Between Pulses The desired time (sec.) between the beginning of the current pulse, and the beginning of the next pulse. The user is responsible for specifying enough time between pulses to allow the CFB data to return to the original baseline after a peak or baseline shift. Reference Offset The calibration heater power (expressed in % of full power) setting for the experiment. Run Data Filename Desired filename for the experimental data. Also contained in the data files (file header) are the run parameters which are used to generate the data, as well as all of the calibration constants for the cell. Other features of the Calibration Setup window (All Pulse Same or Unique Pulses, Execute & Terminate Run, File menu) are identical to those same elements in the Injection Setup window (see Section 5.1) and will not be discussed separately here.

Set Thermostat Temperature Opens the Set Thermostat Temperature window.

Allows user access to the thermostat temperature control window. The current target thermostat temperature is displayed along with the desired thermostat temperature of the jacket. By clicking on the 'Set Temp' button, you will be changing the jacket's target temperature to the value in the 'Desired Thermostat Temperature' text box. Whenever an ITC Cell is in it's idle state (Thermostatting), it will be thermostatting at the current thermostat temperature. Users may change the thermostat temperature via this menu option. If the ITC cell then falls below the specified thermostat temperature, heat will be added to the cell. If the temperature of the cell is above the thermostat temperature, then no heat will be added to the cell, and it will be allowed to drift downward in temperature (through heat leak to the room). This means that a thermostat temperature that is below room temperature will never be achieved without the aid of an external water bath.

-30-


Set Water Bath Opens the ITC Set Water Bath window.

Allows user access to the external bath control window. The current target temperature is displayed along with the desired temperature of the bath. By clicking on the 'Set Bath' button, you will be changing the bath's target temperature to the value in the 'Desired Temperature' text box. The external water bath is used to simulate various ambient temperatures, as the ITC cell must be several degrees above ambient temperature to function properly. Whenever the ITC cell is in the Thermostatting state then the user will be allowed to alter the current bath setting. When the cell is in a non-idle state, the 'Set Bath' button will be disabled to prevent users from throwing a running ITC cell out of equilibration. Unlike the DSC bath control windows, ITC bath control windows do not contain any circulation controls. Any time an external water bath is connected to an ITC Cell and turned on, the bath will be circulating through the ITC cooling plates within the ITC core instrument.

Desiccator On A check mark next to this menu option indicates that the desiccant system is circulating. No check mark indicates that the desiccant system is not circulating. The purpose of the desiccant system is to remove any moisture from the internals of the Cell unit. Desiccation is only available to the user when the ITC cell is in the Thermostatting state. Running the desiccation for about 30 minutes every few days will maintain a dry internal atmosphere for the instrument. More or less frequent desiccation will be required depending on operating temperature ambient humidity.

Terminate Run This option will cause the specified cell to be reset. A run in progress is ended, the injector system returns to home, and the cell enters the thermostatting state. Datafiles for a run which is terminated will be stored in the host computer.

DSC Cell (Cell 1 or Cell 2) Opens the DSC drop down menu.

-31-


Contains all the main menu options for a DSC cell. Provides access to all windows necessary to calibrate the cell, setup and carry out a DSC experiment. Also accesses control of DSC cell peripherals (water bath, desiccator).

DSC Setup Opens the DSC Setup window. Refer to Section 6.1 for a complete description.

Calibrate DSC Opens the DSC Calibration pulse window.

Allows user access to the DSC Calibration window, normally used when a run is in progress. User may turn on and off calibration pulses for a DSC cell. Once a pulse has been turned on, this window may not be closed (it may be minimized) until the pulse has been turned off. Refer to Section 7.2 for a detailed description of how to calibrate your DSC cell.

-32-


Set Water Bath Opens the DSC Set Water Bath window.

Allows user access to the external bath control window. The current target temperature is displayed along with the desired temperature of the bath. By clicking on the 'Set Bath' button, you will be changing the bath's target temperature to the value in the 'Desired Temperature' text box. Additionally, users may control whether or not the bath is being circulated through the jacket. By circulating the bath through the jacket, users may rapidly cool their DSC cell. When a DSC cell is in the Cell Idle or All Scans Complete state then users will have complete control of the bath setting and the circulation on/off. If the DSC cell is in a non-idle state then both the Set Bath Temp and circulation control will be disabled. All bath control is handled by the MCS Control Unit when the DSC cell is not idle.

Desiccator On A check mark next to this menu option indicates that the desiccant system is circulating. Desiccation occurs automatically during the Desiccating state of a DSC run but users may manually control desiccation while the cell is in an idle state (Cell Idle or All Scans Complete).

Terminate Run Runs in progress are aborted and datafiles buffered at the MCS Control Unit for the cell are deleted. The cell enters the idle state.

-33-

Section 5-Running an ITC Experiment

Section 5

Running an ITC Experiment Section Contents: 5.1 Using Observer-ITC 5.2 Designing ITC Experiments 5.3 Sample Preparation 5.4 Cell Loading 5.5 Injection Syringe Filling 5.6 Cell & Syringe Cleaning 5.7 Below Room Temperature Operation 5.8 Far Above Room Temperature Operation 5.9 Precautions 5.10 Troubleshooting 5.11 ITC Experimental Tutorial 5.12 Maximizing Baseline Repeatability 5.13 Selecting the Proper Stirring Rate

-34-

Section 5.1-Using Observer-ITC

Section 5.1

Using Observer-ITC

Pictured above is the ITC setup window. It is here that users will begin all ITC experiments. User defines the run parameters and transmits them to the Control Unit. The setup window is also used to create/edit/save run parameter files (setup files). Once all parameters are as desired, the file can be saved with the File:Save menu option. Once saved, the same run parameters can be loaded (at some later date) into the Setup window with the File:Load menu option. Data files may also be used in the same way in order to quickly set up a new experiment using the same set parameters used for the old data file. When loading a data file into the setup form the run parameters are contained in and extracted from the data file header.

Run Parameters Defined: Number of Injections Total number of injections for a given titration (ITC) experiment. Experimental Temp The desired experimental temperature. Initial Delay Once equilibration is completed, this indicates the time (sec.) into the experiment before beginning the first injection. Volume The desired volume (µl) of titrant for the selected injection(s).

-35-

Section 5.1-Using Observer-ITC Injection Duration The duration (sec.) of the injection. For any given syringe constant and volume the user is provided 25 possible durations to choose from. If the user chooses a new syringe constant from the 'Syringe In Use' listbox then the list of available durations will be updated, and the value yielding an injection rate closest to 0.5 µl/sec. will be chosen for all injections (actual value depends also on the chosen volume for each injection). Likewise, if the user selects a new volume for a particular injection(s), then the list of available durations will be updated for all relevant injections, and again the value yielding an injection rate closest to 0.5 µl/sec. will be chosen for all relevant injections. Time Between Injections The desired time (sec.) between the beginning of the selected injection and the beginning of the next injection. The user is responsible for specifying enough time between injections to allow the CFB system to return to baseline after a peak deflection. Typical values for this parameter range from 120 seconds and up depending on the peak size. Reference Offset Throughout an ITC experiment, a small constant amount of power is continuously supplied to the reference offset heater of the reference cell, which in turn activates the feedback heater on the sample cell. The power selected here (in % of maximum power) must be sufficiently large so that exothermic injections do not cause the feedback power in the sample cell to go below zero. For experiments where exothermic peaks are no larger than 1000 µcal, a setting in the 10-20% range is usually adequate. As the setting is increased to the upper end, you may notice a tendency for the cell temperature to increase very slowly (ca. 0.1 - 0.2 deg/hr) during an experiment so it is best to use the higher settings only when required. Syringe In Use The injection syringe which will be used for the experiment is selected here. Syringes are numbered #1 (50 µl), #2 (100 µl) and #3 (250 µl) and have maximum delivery volumes of ca. 65, 130, and 300 µl, respectively. The calibration constants for each syringe (in µl delivered per inch movement of the plunger) are included, but may be changed by the user using the Calibration Constants window, accessed via the Options main menu selection. Run Data Filename User inputs the desired filename which will contain all generated experimental data. Also contained in the data files (file header) will be the run parameters which were used to generate the data and all of the calibration constants for the cell. t1 This is the initial filter period for data collection during an injection. Data is averaged over the period specified by the filter constant before a data point is generated and stored. For reactions which are "instantaneous", a filter period of 2 seconds is sufficient to obtain enough data points on the peak for accurate integration of the area. For reactions with slow kinetics, the filter period may be increased accordingly to avoid accumulation of excess data points.

-36-

Section 5.1-Using Observer-ITC t2 This is the final filter period for an injection. Data will be filtered using the t1 (see above) filter period until tswitch (see below) seconds have elapsed. The filter period will then switch from t1 to t2. This feature allows users to specify smaller filter periods during peaks (while CFB values may be changing more quickly), and larger filter periods for times when CFB data is more nearly constant. tswitch The time (sec.) into an injection to wait before switching the filter period from t1 to t2. Users may prevent any filter switching throughout an injection by specifying a tswitch value that is greater than the Time Between Injections value for the given injection. Syringe Concentration User inputs the concentration (mM) of the contents of the syringe. This is then stored in the datafile header and used in data analysis calculations. Cell Concentration User inputs the concentration (mM) of the contents of the cell. This is then stored in the datafile header and used in data analysis calculations.

Controls Defined: All Injections Same Selecting this option causes all injections to be assigned the current run parameters for injection #1. Editing the parameter text boxes while this option is selected effects all injections.

Unique Injections Selecting this option does not change any of the run parameters. The injection summary table will become visible at the bottom of the setup window. Editing the parameter text boxes while this option is selected effects only the injection which is currently highlighted in the injection summary table. Likewise, the values displayed in the individual parameter boxes apply only to the injection # which is currently highlighted in the injection summary table.

Remaining Injections Same By selecting this option, users will be assigning the run parameters for the currently highlighted injection (in the injection summary table), to all ensuing injections. Likewise, editing an injection parameter while this option is selected affects the currently highlighted injection (in the injection summary table), as well as all ensuing injections. This option appears only after the Unique Injections option has been selected.

Execute Run This button is used for sending run parameters to the MCS Control Unit, and for initiating the start of ITC experiments. The Execute Run button has a different effect on a Cell depending on the current state. If the cell is idle (in the Thermostatting state), then the run parameters will be transmitted, and the run will begin. If the cell is already in a non-idle state, then the run parameters will be updated and the Cell will continue with it's current run using the newly transmitted run parameters for injections not yet carried out.

-37-


Terminate Run The current run in progress will be terminated, and the Cell will enter the Thermostatting state. Users will be prompted to make sure that they want to terminate the current run.

Close Causes the ITC Setup window to be closed. When the window is reopened, the run parameters will appear as they were the last time the window was open.

Injection Summary Table Whenever Unique Injections is selected in the upper left corner of the setup window, there will be an injection summary table visible at the bottom of the window. You may scroll through the list to view the parameters for each injection. Click on an entry in the list to make that injection # the active injection for editing run parameters. The currently highlighted injection's parameters will be displayed in the parameter textboxes. Any changes to the text box values will affect only the highlighted injection.

Menus Defined: File:Load Opens the Load File window. From here users select a setup file to load into the setup window. This feature allows users to recall frequently used run parameters from disk, rather than having to repeatedly enter them manually.

File:Save Opens the Save File window. From here users may save the current run parameters to disk. If at some later date, the same set of run parameters is desired, they may be loaded from disk into the setup window.

File:Data File Comments Opens data file comments window. From here users may type in any comments that they would like to be appended to the data file header.

-38-


Advanced Options

Battery Use On When a check appears next to this menu option then battery use is enabled at the MCS for power to all nanovolt amplifiers. This is the normal mode of operation which acts to negate any undesirable effects from poor line voltage. Check Starting Temp. When a check mark appears next to this menu option then the experimental temperature will be sought before beginning an experiment. In the case where the user is not concerned with the starting temperature then disabling this option will cause the Seek Init. Temp. state to be skipped during the prerun process and the experimental temperature will be the current temperature of the ITC unit. Equilibration will normally be slightly faster using this latter mode. Injector Enabled When a check mark appears next to this menu option the injector is used during an ITC run. There are some instances where the user may be interested in using the ITC unit without injecting anything into the cell. MicroCal's Solid Particle Insert Cell is an accessory which can be used with the ITC unit, and operates without the use of the injector. By disabling this option, the ITC unit will operate as usual without injections. Stirrer Enabled When a check mark appears next to this menu option, then stirrer will be used during an ITC run. There are some instances where the user may be interested in using the ITC unit without stirring. MicroCal's Solid Particle Insert Cell is an accessory which can be used with the ITC unit, and operates without the use of the stirrer. By disabling this option, the ITC unit will operate as usual without stirring. Auto Start Enabled

-39-

Section 5.1-Using Observer-ITC When a check mark appears next to this menu option, then the ITC experiment will proceed through all of the prerun states without any interaction from the user. Specifically, the MCS Control Unit will 'Seek The Syringe' automatically, when the Equilibrating state is complete. This requires that the Injection Syringe be inserted into the ITC Cell Unit prior to clicking the Execute Run button from the ITC Setup window (you will be prompted to do so). Additionally, the Checking Baseline state will be exited when the baseline has become sufficiently flat, rather than waiting for the user to approve the baseline. The baseline flatness criterion for exiting is a maximum baseline slope on the order of .04 µCal/sec over a 30 second interval. Use Preliminary 1st Injection When a check mark appears next to this menu option, then the ITC injection schedule will include a 'small' preliminary first injection of 2 µl. Once this preliminary injection has been added to the injection summary table in the ITC Setup window, you may edit any of the parameters as you would any other injection. This option is used to overcome some commonly seen problems with first injections. Due to syringe handling and small diffusion effects from prerun stirring, first injection heat-areas have been found to be small. By using this Preliminary 1st Injection, and then deleting the first data point from the binding isotherm, this problem is overcome. The volume of this first injection is included in all concentration calculations, however by deleting the first data point prior to fitting the isotherm, accurate fitting results will be obtained. Auto Degas Enabled When a check mark appears next to this menu option, then automatic degassing of the ITC sample cell will take place immediately upon the injector system finding the injection syringe. The degassing is performed by strobing the stirrer several times at high speeds. This effect tends to cause any air bubbles that may be adhered to the cell walls to be released and float to the top of the sample cell's access tubes. Though this feature will release air bubbles formed during cell loading, the cell loading process should always be followed according to the manual. Set Stirring Speed The default stirring rate is 400 RPM, which has been found to be practical for most applications. If the solution in the sample cell contains suspended particles (e.g. agarose beads), then stronger stirring will be necessary.

Enter the desired stir rate into the text box, and click 'OK' to set the stir rate of the next ITC experiment. Click on cancel to ignore any changes made.

-40-

Section 5.2-Designing ITC Experiments

Section 5.2

Designing ITC Experiments For a ligand X binding to a single set of n identical sites on a macromolecule M, i.e., M + X = MX MX + X = MX2 . . . . . . MXn-1 + X = MXn the single-site binding constant is K

=

[filled sites] ___________________ [empty sites][X]

and ∆Go = —R T lnK = ∆Ho-T ∆So Where ∆Go, ∆ Ho and ∆So are the free energy, enthalpy, and entropy change for single site 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 calorimetry is the only technique capable of defining all of these parameters in a single experiment resulting in nearly complete thermodynamic characterization of the interaction. Measuring the binding isotherm at a second temperature allows additional determination of the change in heat capacity of binding through the relation: ∆Cp

=

∆Ho T2 - ∆HoT2 ___________________________ T2 - T1

It is well-known that ∆Cp is a good indicator of changes in hydrophobic interactions with binding, being negative if hydrophobic bonds are formed and positive if they are broken. The critical parameter which determines the shape of the binding isotherm is the unitless constant c, which is the product of the binding constant K times the total macromolecular concentration in the cell at the start of the experiment, Mtot , times the stoichiometry parameter, n. 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

-41-

Section 5.2-Designing ITC Experiments 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 macromolecular 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 ITC sensitivity is ca. 0.5 µcal so for precise measurement each injection should have an average of at least 5-10 µcal of heat absorbed or evolved into the 1.4 ml cell. How these factors impinge on your choice of macromolecular 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 10-6 < Mtot -3. < 10 It requires at least 10 separate injections to define the total binding isotherms and each injection must average ca. 10 µcal, so the total heat Q required in the 1.4 ml cell is 100 µcal, i.e., Q = 100 x 10-6 cal = (15000 cal/mole) (Mtot moles/l ) (1.4 x 10-3 l ) Solving this equation for Mtot gives a minimum concentration of ca. 5 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 5 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 5 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.)

-42-

Section 5.2-Designing ITC Experiments

For example, a 10 µl injection of a 7 x 10-5 M ligand solution would give the minimal 10 µcal 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 which syringe 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. 1.4 ml), and n is the ligand/macromolecule stoichiometry. For cases where n=1, the ligand concentration, Xtot,,should be ca. 42 times Mtot (50 µl syringe), 21 times Mtot (100 µl syringe) or 8.5 times Mtot (250 µl 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. Concerning the latter, if you are doing a total binding isotherm using the 100 µl syringe, then the initial ligand concentration in the syringe must be ca. 20 times larger than the concentration of macromolecule sites in the cell. This can lead to ligand solubility problems, especially if the ligand is another macromolecule. Thus, the concentration of macromolecule in the cell could be chosen close to the low end of the available range, and/or the 250 µl syringe could be used instead of the 100 µl syringe to overcome this problem. 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 µl injection followed by ten 10 µl 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.

-43-

Section 5.3-Sample Preparation

Section 5.3

Sample Preparation 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 diaphragm pump (ca. 27" Hg vacuum) and plastic vacuum chamber placed atop a magnetic stirrer may be used for degassing without excessive boiling. Normally only 3-4 minutes of vacuum is required to degas if the sample is being stirred but much longer without stirring. 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.

-44-

Section 5.4-Cell Loading

Section 5.4

Cell Loading The cells are filled using the long needled 2.5 ml glass syringe, filling from the bottom of the cell to the top. The top of the access tubes are visible in the floor of the injection system. The sample cell is in the center and the reference is offset to the left. Fill slowly until liquid can be seen overflowing the access tube. There is a tendency for a small bubble to be left near the top of the cell. Finish the filling by adding an abrupt spurt of about .25 mL to dislodge any bubbles. Fill the reference cell with water. There is no need to refill the ITC reference with each experiment. A water reference may be good for a week or two with no attention if the water was thoroughly degassed before filling. Add an antibacterial agent (e.g. 0.1% aqueous sodium azide) to the reference cell to prolong the refill period. A small stopper and insertion device has been provided to cap the reference cell access tube after filling, if desired. Remove any excess solution from the floor of the reservoir after filling, following withdrawal of the filling syringe.

-45-

Section 5.5-Injection Syringe Filling

Section 5.5

Injection Syringe Filling The syringe capillary must be completely dry before filling (e.g., methanol rinse followed by 5 minute purge with dry air) or well rinsed with the ligand solution. It is very important to load the injection syringe with the complete absence of bubbles. You will fill the injection syringe while it is in the plastic syringe holder, and your filling configuration should be as pictured below.

After seating the syringe into the syringe holder, the black disk at the top of the syringe should be firmly seated against the surface of the syringe holder, since this controls the final distance between the stir paddle and the bottom of the cell. This

-46-

Section 5.5-Injection Syringe Filling black disk has been positioned correctly, and secured with the set screw, by MicroCal. If you need to reposition this, see the Troubleshooting section.

The syringe's plunger is pulled up to the high edge of the metal filling port located near the top of the syringe. A second syringe is attached to the filling port with a short length of thin plastic tubing. This is used to suction solution into the injection syringe, removing all of the air between the plunger and the solution. Once the solution begins to exit the Filling Port, the syringe plunger is depressed below the filling port. The plastic tubing may now be removed from the filling port. Depress the plunger sharply with the stirring paddle still immersed in sample and then draw solution slowly back up. Repeat this procedure. This insures that any small bubbles formed in loading will be expelled out of the long needle. After filling, rinse the tip of the syringe lightly with water from a washbottle and seat it back into the Syringe Stand. A syringe loaded with titrant must not be flexed or titrant will be displaced from the needle, resulting in an injection volume error for the first injection.

-47-

Section 5.6-Cell and Syringe Cleaning

Section 5.6

Cell and Syringe Cleaning The ITC reference cell doesn't require special cleaning but should be rinsed occasionally using long needle syringes. The ITC sample cell need 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 solids which cannot be removed by rinsing will adhere to the cell interior and these can cause baseline problems if not removed periodically by a thorough cleaning with detergent solution. A cell cleaning apparatus is provided for that purpose.

The device is inserted into the sample cell and seals in the sample cell overflow reservoir in the floor of the injection system. A vacuum trap beaker is connected to the output and a beaker of cleaning solution to the input of the cell cleaning apparatus. A hot solution (~75 deg. C) of 25% Top Job detergent is recommended. Once assembled as above, the vacuum is activated. Allow ca. 200-400 ml of the detergent solution to flow through the cell and then remove the tubing from the detergent solution. Rinse the end of the tubing with a wash bottle to remove all traces of the detergent solution. Then dip the tubing into another beaker containing rinse water and allow ca. 200 ml of flow before removing the tubing from the beaker. Remove the cell cleaning apparatus from the cell, and drain remaining water from the cell using the long-needled 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 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 1.5 - 2% if you measured concentration before the sample was introduced into the cell. Injection syringes may be cleaned with detergent solution and rinsed with water using the same method as described earlier for filling the injection syringe by drawing liquid upward through it's filling port and into the plastic syringe. If you have sufficient ligand solution for rinsing the syringe following the water rinse, then it is recommended before final filling. If you do not have extra ligand solution for rinsing, then follow the water rinse with a methanol rinse and dry the syringe of methanol by purging with air. If any methanol is left in the syringe before filling with ligand solution, then your experiment will be unsuccessful since the heat of dilution of aqueous methanol into water is very large. In the event of clogging, small cleaning wires have been provided for cleaning solids from the injection ports of the stir paddle.

-48-

Section 5.6-Cell and Syringe Cleaning WARNING: The cells of the ITC are fabricated from Hastellloy C, and should never be cleaned with strong acids of any kind. Please consult the booklet provided on corrosion resistance of Hastelloy C before using any substances which have corrosive properties.

-49-

Section 5.7-Below Room Temperature Operation

Section 5.7

Below Room Temperature Operation Since heat can only be added to the cells and jacket of the ITC (i.e., there is no direct way to cool these components), the operational temperature for an experiment must be at least several degrees higher than "ambient temperature". The purpose of the refrigerated circulating bath is to bring about a decrease in the effective ambient temperature to permit studies below actual room temperature. Coolant from this bath circulates through metal plates which surround the cell and jacket assembly. It is important to realize that about 2.5 inches of polyurethane foam insulation are sandwiched between the metal plates and the jacket, which makes heat transfer between the plates and jacket very slow. In fact, once the temperature of the circulating bath is changed, it requires ca. 8-10 hours for equilibration to take place again and achieve a stable baseline. Because of this slow equilibration, the most time-efficient way to conduct a series of experiments at different operating temperatures (some of which are below room temperature) is to avoid changing the temperature of the circulating bath whenever possible. For example, if you wish to carry out titration experiments at 10 C and 25 C in one day, then the best way to do this would be to set the temperature of the circulating bath at 5 C and set the ITC thermostatting temperature to 10 C before leaving the laboratory in the evening. When you arrive the next morning, everything is equilibrated for the experiment at 10 C. After completing that experiment, set the ITC for a second experiment at 25 C but do not change the set temperature of the circulating bath. It will only require ca. 20 minutes for the ITC to scan from 10 C to 25 C and equilibrate at the new temperature. Thus, by working progressively from experiments at the low temperatures to experiments at the high temperatures without changing the temperature of the circulating bath, it is possible to do many experiments at different temperatures in the same day. There are some limitations to this procedure. There tends to be a gradual degradation in baseline quality as the ITC operating temperature moves further and further above the temperature of the circulating coolant. For experiments where maximum performance of the ITC is required, it is best not to do experiments at temperatures which are more than ca. 35 C higher than the temperature of the circulating coolant.

-50-

Section 5.8-Above Room Temperature Operation

Section 5.8

Far Above Room Temperature Operation For the reasons mentioned in the previous section, operating far above room temperature also requires an awareness of the possibility of baseline degradation if maximum performance is needed. Thus, if you wish to carry out an experiment at 55 C, better performance would result if the ITC was first equilibrated overnight with the bath circulator set at 50 C as opposed to simply scanning the ITC up to 55 C with the bath not circulating.

Thermostatting the Injection Syringe: You will notice that there are two copper connectors on the rear of the injection tower of the ITC. Plastic tubing may be connected to these in order to circulate coolant from an external bath, so that the syringe and contents may be thermostatted at the same or a different temperature where the cell is thermostatted. This might be useful, for example, if you are making injections of a component which is labile at room temperature. It should be added that there is no need to routinely thermostat the syringe to the same temperature as the sample cell in order to avoid heat effects during injections which arise from such temperature differences. The fluid from the syringe is automatically brought to cell temperature before injection, no matter what the temperature of the syringe.

-51-

Section 5.9-Precautions

Section 5.9

Precautions The stirring syringe needles must not be bent even slightly or sensitivity will be compromised. The syringe plungers must also not be bent since the injection system must be able to find the plunger top. Exercise care at all times in the handling of the stirring syringes. Carefully remove the syringe from the instrument after an experiment ends, clean, dry, and store safely.

-52-

Section 5.10-Troubleshooting

Section 5.10

Troubleshooting Problems may take the form of a short term noise in the baseline, long term drift of the baseline, and erratic peak sizes. Mistakes in technique can cause all of these problems. Problems are most often due to either cell/syringe filling or stirring. PROBLEM: Instrument never leaves the Seek Initial Temperature state. SOLUTION: Since the ITC cells and jacket cannot be cooled directly, experiments must be carried out at temperatures above the effective ambient temperature. If you try to do otherwise, Observer will stall in the Seek Initial Temperature state. When you are not circulating coolant through the cooling plates, your experimental temperature must be at least 4 deg C. above room temperature, to allow for heat generated within the ITC unit. When operating below room temperature and flowing coolant from the circulating bath, then the lowest experimental temperature you may use is 4-5 deg C. above the bath temperature. PROBLEM: Prior to inserting the injection syringe the baseline is not stable (normal peak to peak noise ~ .025 µCal/sec, drift < .5 µCal/sec/hour). SOLUTION: Higher than normal drift may indicate the need for desiccation. Desiccate for one hour and repeat. Higher than normal drift is expected for 12 hours after changing the bath set temperature. Higher than normal drift is expected after increasing the operating temperature. The drift is higher for larger temperature changes. The drift will decrease to zero with time. Your ITC instrument is responsive to changes in room temperature, but because of the thick thermal insulation which separates the jacket from the room it responds very slowly to these temperature changes. Thus, the normal 10 min. on/off cycle of an air conditioner or heating system has little or no effect on the baseline so long as the average temperature remains constant. However, if the room where the calorimeter is located increases in temperature by several degrees beginning at the start of each working day, then the baseline will slowly drift for many hours before it levels out and becomes flat. The total change in baseline over this long time period will amount to ca. 0.05 mcal/sec for each degree change in room temperature. This effect may be dampened to some extent, but not eliminated, by always circulating coolant through the cooling plates. The best solution is to make sure the room is thermostatted at the same temperature for 24 hours a day while the instrument is being used. PROBLEM: Baseline is stable without stirring, but becomes unstable after syringe is inserted and stirring begins prior to injections.

-53-

Section 5.10-Troubleshooting SOLUTION: Indicates a stirring problem. May be debris or bubbles in cell, bent injection syringe needle, improper vertical positioning of the syringe, 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 ITC unit on a suitable stand and flushing the sample cell with the filling syringe. The injection system must not be activated in any way during this procedure. If your test samples are suspensions of particles try higher stirring rate to achieve uniform suspension. Bent injection syringe needle - test by rolling on flat table top. Bent needles can sometimes be straightened by a machinist using a lathe. Improper injection syringe vertical position - with the syringe inserted in the instrument loosen the set screw on black plastic disk on the top of the injection syringe glass and slide up slightly. Hold the syringe holder firmly with one hand and rotate the injection syringe glass so that it may be pushed up and down within its holder. Slowly push the syringe through the holder until the bottom of the cell is felt as an impediment. Raise the position of the syringe .1 inches, push the black disk down to the top of the holder and retighten the set screw. Injection System Alignment - The injection system is gimbal spring mounted to permit precise angular alignment with the sample cell access tube. The three lock nuts on the baseplate of the injection system set this alignment. Unless these are known to have been adjusted since the instrument was purchased they shouldn't be adjusted. Set the alignment by first emptying the sample cell then inserting the alignment device. By viewing the device from directly overhead small angular misalignments can be seen and corrected with small adjustments to the lock nuts. Access the two side lock nuts by removing the rear plate from the injection system and sliding the side plates out. PROBLEM: Titration data shows one or several peaks smaller than the apparent data peak envelope. SOLUTION: Likely attributable to bubbles in the injection syringe. Prepare degassed solutions and repeat following loading instructions for the injection syringe.

-54-

Section 5.11-ITC Experimental Tutorial

Section 5.11

ITC Experimental Tutorial The following experimental tutorial is designed to acquaint you with the basic features of both the hardware and software of the ITC instrument, as well as to provide experience with several manipulations which must be mastered in order to get the highest quality data from your instrument. Rather than beginning experimentation on the ITC using precious biological samples, we strongly suggest that each user of the ITC instrument complete the following tutorial first, using sucrose solutions, so that irreplaceable samples are not wasted while mastering the appropriate techniques. At this point, your instrument should have been completely assembled according to the directions provided in Section 2. The black anodized aluminum injection system should have been correctly mounted on the top surface of the ITC cell assembly, all electrical connections made, and all MicroCal software installed on the hard drive of your computer (this was done for you if you purchased your host computer from MicroCal). The recommended procedures for preparation of solutions, degassing, filling the cell and injection syringe, etc. have already been discussed in Section 5 and the following tutorial assumes your familiarity with those procedures.

I. Sample Preparation Begin by degassing (with stirring) ca. 100 ml of distilled water for ca. 5 minutes using your Gast diaphragm pump (or suitable substitute) and degassing apparatus. This degassed water will be the solution which will go into the sample cell (and also reference cell if it does not already contain water). Use ca. 15 ml of the degassed water in a test tube and add sucrose to make a 5% sucrose solution, using stirring to be sure all sucrose has dissolved. This solution will be loaded into the injection syringe later.

II. Filling The Cells The Cell Entry Port consists of a 3/4" hole in the center of the black, fluted housing which encloses the stirring motor on top of the ITC Cell Unit. At the bottom of the port, the entry tube to the sample cell is in the center and the reference cell is off to the left side (If overhead lighting is poor, you may want to examine these using a flashlight). Load the 2.5 ml glass filling syringe (8" needle) with the degassed water, and tap the syringe bottom after loading so all bubbles float to the top surface. Enter the needle into the sample cell entry tube and carefully slide the needle down the tube until it just touches the bottom of the cell. Begin to slowly depress the plunger so the cell fills from the bottom up. After ca. 1.5 ml of degassed water has been entered, you will be able to see the water come to the top of the small entry tube. Once you see the liquid level, raise the syringe a millimeter or so off the bottom of the cell and depress the plunger very quickly to deliver a spurt of ca. .3 ml. The purpose of the last spurt is to dislodge any bubbles which might be clinging where the entry tube joins the cell. If the reference cell has not previously been filled with water, then carry out the same procedure to fill it. There is a small cap and positioning tool which has been provided that you may use to close off the reference cell if you desire to do this.

III. Assembling and Filling The Injection Syringe Locate the 100 µl (medium bore) injection syringe with the attached long needle that has a stir paddle at it's tip. Locate the white plastic (delrin) syringe holder. The syringe holder is expanded at the top with an O ring immediately below the expanded portion. Carefully slide the end of the long syringe needle into the top hole and out the bottom hole of the syringe holder as shown in the diagram below. Push carefully on the black plastic restraint ring at the top of the syringe until it seats firmly against the top delrin surface of the syringe holder. (The position of the restraint ring has been fixed prior to shipment so that the stir paddle will be located at the correct height in the sample cell after it has been seated into the barrel of the injection system.)

-55-


Locate the clear plexiglas syringe stand. Seat the assembled syringe/syringe holder assembly into the center of the syringe stand, as shown below.

You are now ready to fill the injection syringe with the sucrose solution you have prepared. Begin by raising the injection syringe plunger to the high edge of the metal filling port located near the top of the syringe, as shown at the top of the picture below. Now, raise the injection syringe/holder assembly slightly so that you can slide the test tube of sucrose solution underneath the tip of the injection syringe's stir paddle. While holding the test tube with one hand, lower the injection syringe/syringe holder assembly until it is again seated in the plexiglas syringe stand. The injection syringe's stir paddle should now be completely submersed into the sucrose solution. Now locate the clear plastic filling syringe and the length of clear plastic tubing which came with your instrument. Insert one end of the tubing over the tip of the filling syringe, and the other end over the injection syringe's metal filling port. Your setup should now be as shown below.

-56-


By slowly withdrawing the plunger of the plastic filling syringe, you will draw the sucrose solution into the injection syringe. Once the solution begins to exit the top filling port, depress the plunger of the injection syringe until it is slightly below the side-arm filling port, thereby stopping the flow of the sucrose solution. Remove the plastic tubing from the side-arm filling port. With the stir paddle of the injection syringe still immersed in the sucrose solution, depress the plunger sharply and draw it back slowly while being sure not to let the plunger come all the way up to the side-arm filling port. Repeat the procedure a second time to insure that any small bubbles, which might have lodged in the long needle of the injection syringe during filling, are expelled into the excess sucrose solution. Your injection syringe is now filled. Carefully raise the Syringe/Holder Assembly out of the Syringe Stand, remove the vial containing the excess sucrose solution, rinse the stir paddle and lower part of the needle with a wash bottle, and reseat the Syringe/Holder Assembly onto the Syringe Stand. Anytime that you are handling the injection syringe after it has been filled with titrant solution, you must be careful not to bend the long needle since this will cause some solution to be expelled from the syringe and will result in a poor first injection for your experiment. Unlike the present experiment where we are using sucrose solution, there may be times when the titrant you are using is very valuable. Small culture tubes have been provided which minimize the volume of titrant required to load the filling syringe. It must be recognized however that liquid transfer into and out of the injection syringe occurs through a tiny hole about 1 cm above the bottom of the stir paddle, which must then always be kept below the liquid surface in the culture tube.

IV. Preliminary Operations Using Observer Once your instrument is up and running routinely, we recommend that power to the Control Unit be left on continuously for best performance. For this tutorial however, we want to begin with a power down-power up situation to illustrate a point. Using the AC power switch on the rear of the Control Unit, turn the control unit off and leave it off until prompted by Observer to turn it on. At the host computer terminal, double-click on the MCS Observer icon located in the Windows Program Manager. A large window displaying MicroCal's logo will appear. After opening a serial port for MCS communications, the Observer will try to communicate with the Control Unit. Because the power has been turned off, memory at the on-board computer in the Control Unit has been cleared so its software operating system, MCS OS, is not available to establish contact with Observer at the host computer. When contact cannot be made by Observer, the following screen appears.

-57-


Click on the 'Download MCS OS' option. Wait for the prompt to turn on the Control Unit's power. Failure to do so will prevent the file transfer from executing. Once prompted to power up the MCS Control Unit, doing so will initiate the download process. You will see a % complete indicator increasing, until all files have been received. After approximately a 20 second delay (while files are decompressed at the Control Unit), Observer will again attempt to get the Cell-Configuration information from the Controller, this time successfully. Observer will configure itself according to the number and types of calorimetric units included in your system, and the main window of Observer will appear. Additionally, Origin will be running in the lower right corner of the screen. You screen should now appear as pictured below.

-58-


By observing the data channel displays on the main window of Observer, you can verify that data is continuously being received from the Control Unit. If you see the values in the text boxes changing approximately every 5 seconds (though only slightly) then you are ready to continue with this tutorial. We will start by getting the screen display of the host computer such that we can easily see everything that is going on. First, move the Observer main window to the upper left area of the video screen. Now, under the Options menu, click on Cell Display so that the three submenus appear: Display Cell 1, Display Cell 2 and Display Both Cells. Click on the appropriate one to display only the data from the ITC cell we are working with. If you only have one cell then you may ignore this step. We now should have just one cell's data channels being displayed. Confirm that we have the correct cell displayed by seeing that the cell status text box reads Thermostatting ITC. Users should note that any time only one cell is displayed, by double clicking on the Cell Status text box the display will toggle to the other cell's data. Second, move the Origin window to the upper right most area of the screen. Put the mouse cursor on top of the lower left corner of the main Origin window so that the cursor becomes a double arrow. Now click the left mouse button and while holding the button down, drag the corner of the window so that Origin's main window occupies most of the video screen. Do not make the Origin window so big that you can no longer see the MCS Observer. Now click on the maximize button (upward arrow in the upper right corner) of the appropriate Origin plot window (not the main Origin window) so that it occupies the entire main Origin window. You now should be able to see both the MCS Observer as well as the Origin plot window where your data will be plotted, as shown below.

-59-


Check the cell status box for your ITC cell. The status box should read Thermostatting ITC. If it doesn't, then from the main ITC menu (Cell 1 or Cell 2 menu option) click on Terminate Run, and click on the Yes button when prompted. After a slight delay the cell will go into the Thermostatting ITC state.

V. Setting Run Parameters From the main ITC menu select ITC Setup and the ITC setup window will appear. This is where all the run parameters are defined for an ITC run. In the upper left corner, click on All Injections Same. The button to the left of the All Injections Same text should be blackened indicating that this is the active choice. Now, working from top to bottom in the text boxes, we will define the experimental parameters. -Enter 11 in the # Injections text box -Enter 30 in the Experimental Temperature text box. -Enter 60 in the Initial Delay text box. -Enter 10 in the Volume text box. -Leave the default Duration value that is displayed (will be ca. twice the volume) in the Duration list box. -Enter 210 in the Time Between Injections text box. -Enter 10 in the Exothermic Headroom text box. -In the Syringe In Use list box click once on the downward arrow. A list of three syringes will drop down. Click once on the 43 µl/in list entry. You may notice that the duration value in the Duration list box changed slightly. That's normal, and you may use the new value if it has in fact changed. -Type sucr1.itc in the Run Data Filename text box. You need not type the filename extension, however since regardless of what extension is typed in this text box the datafile extension will be .itc. -Now going to the top right area of the setup window, enter 2 in the T1 text box. -Enter 2 in the T2 text box. -Enter 210 in the T Switch text box.

-60-

Section 5.11-ITC Experimental Tutorial Since we are not actually working with real biological samples for the tutorial, you may leave the Concentrations In Syringe and In Cell as they are. When working with samples be sure and put the appropriate concentrations for the syringe and cell contents into these text boxes, as they will be used during data analysis calculations in Origin. By filling in the ITC Injection Setup as described above, you have indicated that you will be using the medium-sized syringe (delivers 43 µl for each inch the plunger is depressed) to deliver 11 identical injections of 10 µl each, with the first being made 60 sec into the experiment and each successive injection spaced at 210 sec following the previous injection. Since t1 and t2 are both 2 sec, all data will be averaged for 2 sec before a data point is stored, which will lead to a total of 1185 data points. This experiment will be automatically carried out at 30 C. It is expected that the amount of heat from each injection will not be exceedingly large, so that 10% exothermic headroom should be more than ample. Click once on the Advanced Options menu. A drop down menu with eight submenus will appear.

The top seven menus simply work with check marks. All four of the top sub menus will have check marks next to them indicating that the options are active, while the bottom three options will not be active indicated by the absence of check marks. Click once on the top most submenu, Battery Use On. You should see that the Battery Use On submenu option no longer has a check mark next to it. Click once on the Battery Use On submenu option again to turn the option back to active. This is how the top seven advanced options submenus work. Now, click once on the Set Stirring Speed submenu. A small window will appear with a text box to enter the desired stirring speed. Enter 410 into the Desired Stir Rate text box, and click OK to accept the change. The window will disappear, and you will be back in the ITC setup window. If you had clicked on Cancel instead of OK then any changes to the desired stir rate would have been ignored. Now, click on the File menu , and then on the Data File Comments submenu. The Data File Comments window will open.

-61-


You may type in any comments that you want to be added to the data file header. Now click on the File menu , and then on the Save File submenu. The Save File window will appear.

Type in sucr1.inj and click on the OK button. You now have saved these ITC run parameters to disk and they are available to you for later use. By default, all setup files like the one that you just saved will reside in the c:\mcs\setup subdirectory. Data file comments are not saved to the setup files since they are usually unique to each experiment. To demonstrate the reuse of setup files do the following: Enter 5 in the # Injections text box. Now click on Unique Injections. An injection summary table will appear on the bottom of the ITC Setup Window, displaying 5 potentially unique injections. Now click on the Load File submenu. The Load File window will appear, and all of the ITC setup files (.inj extension) will be listed on the left side.

-62-


Click on the sucr1.inj entry that you just created and choose 'OK. The parameters from this file will appear in the ITC Setup Window. Confirm that the # Injections text box again displays 10, and that All Injection Same is chosen in the upper left corner. The injection summary table that appeared at the bottom of the window should no longer be visible.

VI. Beginning The Experiment Now let's begin the ITC experiment. Click once on the Execute Run button found on the left side of the setup window. A small window will appear on top of the setup window. Follow along with the messages as the run parameters are transmitted to the MCS Control Unit, and the run is started. You should now click on the 'Close' button on the left side of the setup window (or you may minimize the window by clicking on the upper right most down-arrow of the window). The ITC setup window will no longer be visible (or will be minimized). It should be realized that windows are best to be closed when you are finished using then, rather than simply minimized. Open windows exhaust system resources, and thus should be closed when no longer needed. By looking at the appropriate Cell Status box of the main window (bottom most text box), you can follow along with ITC cell as it progresses through several prerun states. It should now be in the Seek Init. Temp. state, with the temperature linearly increasing towards 30 degrees.

-63-


VII. Manipulating Origin If you look at the appropriate plot window in Origin, you can watch the prerun data being plotted versus time. The legend at the top of the plot window indicates the colors of the different data channels being plotted. The blue dataset in layer 1 is the Cell Feedback, the red dataset in layer 2 is the Jacket Feedback, and the black dataset in layer 3 is the jacket temperature. The units of each dataset can be found directly above their respective axes.

You can see that there are three different Y-axis in this plot window. On the rightmost Y axis, click and drag the bottom slide bar to 22 and the upper slide bar to 32. Now click on the Zoom button located in the bottom right region of the plot window. The black dataset (Temperature) should now be displayed on a scale from 22 to 32. You may zoom in on any layer by following these steps in the appropriate layer. The legend at the top of the plot window, along with the axis label colors indicate which dataset is plotted in which layer. If you would like to zoom out on a plotted layer, then move the slide bars beyond the Y axis minimum and maximum values for the appropriate layer (we are working in layer 3).

-64-


Now by clicking on the 'Zoom' button, layer 3 will zoom out. The zoom out factor is found behind the 'Values' button. We will discuss the 'Values' button immediately below. Demonstration of Auto Scroll/Scale Feature: Click once on the Origin control labeled 'Values' in the lower right area of the plot window. A simple dialog box will open.

-65-

Section 5.11-ITC Experimental Tutorial The first entry in this dialog box is the zoom out scale factor. As you can see, the factor is set to 2, which means that a zoom out will result in twice the Y axis range, centered about the same point as is the current scale. You may set this value directly from this text box. The checkbox labeled 'DDE Scale or Scroll?' allows you to turn on/off the auto scroll/scale feature. When the box is checked then the feature is active. By clicking on the 'Which?' listbox, you may change the selection to either Scale or Scroll. When Scale is selected (and active), whenever the displayed data exceeds the current X Axis display, then the X Axis will be scaled by the factor appearing in the 'Fraction of Full Scale?' text box. Likewise, when Scroll is selected (and active), whenever the displayed data exceeds the current X Axis display, then the X Axis will scroll by the factor appearing in the 'Fraction of Full Scale?' text box. Finally, the 'First DDE on Screen' check box indicates whether or not the data display will be scaled or scrolled relative to the first data point that is received. Users will probably want this option left as active. When the temperature reaches 30 degrees (black dataset in layer 3), the ITC cell will exit the Seek Init Temp state and will enter the Equilibrating state. When the Delta-T signal (red dataset in layer 2) becomes positive and the CFB signal becomes positive and flat, then the Equilibration state will be exited.

VIII. Inserting The Injection Syringe The ITC cell will then be in the 'Ready For Syringe' state, which indicates that the cell is ready to accept the injection syringe. There will be a button located just below the cell status text box.

Carefully remove the Syringe/Holder Assembly from the plexiglas Syringe Stand and insert the stir paddle end of the needle into the entry port for the sample cell, located in the center of the Cell Port Entry. Lower the syringe slowly until the O ring touches the top of the Cell Port Entry. Push firmly on the top of the Assembly (away from the syringe) until the O ring goes all the way into the Cell Port Entry hole. In carrying out the procedure described above, it is important to do it with no flexing of the syringe needle, since if it bends during insertion then liquid will be forced out of the syringe and the volume delivered in the first injection will be incorrect. Once you have inserted the injection syringe into the ITC cell, then you must click on the Syringe Inserted button to initiate the seeking of the syringe.

-66-


After clicking on the Syringe Inserted button, the injector will find the plunger of the injector syringe. Once it finds it, it will clamp it securely, turn on the stir motor, and send a message to Observer indicating the available volume of titrant in the syringe. If by chance there was no syringe inserted, then the injector system would return to its Home position, return an error message to the interface, and the run would be aborted.

IX. Baseline Equilibration When the syringe is successfully grasped the Baseline OK button will appear as shown below, and only the CFB data in Origin will be seen in the plot window.

The CFB data is the differential power baseline, and it must flatten out and equilibrate before the injections are started. Turning on the stirrer has caused a disturbance in the CFB but it will stabilize in a few minutes. The initial Y axis scale for CFB may not be sensitive enough to allow you to determine when equilibration is complete. Once the CFB data appears to be flat on this scale, we will need to zoom in to get a better look at the data. You could use the slide bars to zoom in, as you did earlier. Alternatively, as we will do here, you may double-click on the Y axis line, which brings the Y Axis Dialog Box into view. In the upper left corner of this dialog box, you will see Y axis limit boxes designated as From: and To: The From: box is darkened and ready to edit. Note the present CFB reading in the appropriate Observer data box. Subtract 1 µcal/sec from that reading and enter the resulting number in the From: box. Press the tab key on your keyboard, which then darkens the To: box for editing. Enter a number in here which is 1 µcal/sec larger than the current CFB reading. Click on OK and the dialog box closes. Now your data plot should have full scale of 2 µcal/sec on the Y-axis and the current CFB data should be centered in the plot. The CFB signal should now be visibly leveling off at this Y-axis scale expansion. The longer you wait, the flatter it will become. Once the CFB signal is changing by ca. 0.1 µcal/sec, or less, over a five minute period then click on the Baseline OK button in the Observer window. Refer to the ITC Troubleshooting section for a description of problems which may cause unstable baselines, or unusually long baseline equilibration periods.

X. Data Collection The ITC cell will now begin it's initial delay. Within a short time, a new Origin plot window will appear, and all ensuing experimental data will be plotted in it. After the 60 second initial delay, the first injection will be made. You can follow the injection process by watching the Cell Status text box. When the injection is being made, when it has finished, and the data collection period for the injection are all indicated in the Cell Status text box. Anytime after the syringe has been grasped, users have access to the current titrant volume in the syringe. This value is displayed in the ITC Setup window along with the current syringe in use.

-67-


If users decide after beginning an experiment that they would like to increase the number of injections or change other run parameters, then by referencing the 'Volume In Syringe' value, it is known how much more volume may be injected for the current experiment. The injector system will not inject more than the available volume, regardless of the user's requested volume. In other experiments where you might wish to change run parameters for a run in progress, simply edit the appropriate parameter boxes in the ITC Injection Setup table and then click on Execute Run and the new parameters will be used for the remainder of the run. After all 11 injections are completed, the data file can be found on your hard drive in the \MCS\DATA subdirectory. The injector system will return to its Home position and the run is finished. Carefully remove the Syringe/Holder Assembly from its seated position in the Cell Entry Port and transfer it to its cradle on the Syringe Stand. You will now have the experimental data displayed in Origin. If you wish, double click on the X axis and enter -200 and 2700 in the From: and To: boxes so all of your data will be displayed (you may instead choose the Plot:Rescale To Show All menu option). Pictured below is data from a similar sucrose dilution experiment (4.5% Sucrose) generated at MicroCal.

XI. Doing The Control Experiment Now that data has been obtained for injections of 5% sucrose into water, you should proceed to do the control experiment for 11 equivalent injections of 5% sucrose into 5% sucrose. Since the control experiment involves identical solutions in this case, any heat effects which you see will be the result of very slight temperature mismatch between the solution in the cell

-68-

Section 5.11-ITC Experimental Tutorial and the solution entering the cell from the syringe. Once this control data is subtracted from the original sucrose-into-water data, the difference will be due strictly to the heat of dilution of sucrose. It should be added that normal control experiments for ligand binding to macromolecule involve injections of the ligand solution into buffer which contains no macromolecule. In these cases, the two solutions are non-identical and control peaks will include effects from heat of dilution of ligand as well as those due to slight temperature mismatch. Drain the solution from the sample cell, rinse twice with 5% sucrose and finally fill with 5% sucrose. Refill the syringe to its full position with 5% sucrose. You are now ready to do the control experiment according to the same procedures you used for the sample experiment. Name your data file sucr1ctl.itc for this control experiment.

-69-


XII. Plotting and Analyzing Data In Origin At this point you should have completed two ITC experiments, the original 5% sucrose into water experiment as well as the 5% sucrose into 5% sucrose control experiment. We can now go to Origin to analyze the data. Click on the Origin-ITC Data Analysis icon in Program Manager. You should see the ITC raw data template as pictured below.

If this is not what you see, then click once on the Origin 'Edit' menu, and then again on the 'Change Menu'\'ITC Data Analysis' submenu. You may answer yes if prompted to start a new session. You now will be looking at the Raw ITC data template. To import your MCS data into the plot window, click on the 'Read (ITC)MCS Data' button. Find your way to the \MCS\DATA subdirectory, select the sucr1.itc entry and click 'OK'. Origin will automatically plot the raw data, generate a baseline for the raw data, integrate all peaks, and display the integration results in the Delta H window. We will discuss the Delta H window in some detail later on. Click on the Raw ITC Data window to make it the active window (or you may select RawITC from the Window menu in Origin). Follow the same procedure as in the previous step to read in the control data (select the control datafile sucrctl.itc this time instead of the sucr1.itc datafile). Though Origin now has the raw data from both the experiment and the control, only the control data is displayed in the plot window. Locate the layer icon (a gray box with '1' appearing in it) in the upper left corner of the Raw ITC Data window, and double click on it. The layer control dialog box will appear.

-70-


You can see that the layer contents contains two datasets, sucr1ctl.raw_cp and sucr1ctl.base. Directly to the left is the available data, and by highlighting the desired dataset, and clicking the => button you will move the sucr1.raw_cp and sucr1.base datasets into the layer contents. After doing so, the layer dialog box should appear as pictured below.

-71-

Section 5.11-ITC Experimental Tutorial When you have all four datasets included in the layer contents list, click 'OK'. You will return to the Raw ITC Data window, and you should see both raw datasets and their baselines plotted. To gauge the relative success (or failure) of your experiment you can compare your raw data to the plot below.

These data were generated at MicroCal on an MCS ITC instrument using a 4.5% sucrose solution and the same run parameters used in this tutorial. Your peak sizes may differ a little (peak size is proportional to ca. the 1.5th power of sucrose concentration). The size of control peaks will also vary a little from instrument to instrument and with actual room temperature. Note also that the above control peaks show an initial exothermic swing followed by a slightly larger endothermic swing. This updown swing within the same injection peak is not unusual to see for peaks with extremely small net area, but you may or may not see it with your data. Now click on the DeltaH plot window in Origin or select DeltaH from the Window menu of Origin. Origin automatically plots concentration-normalized areas (kcal per mole of injectant, designated with an .ndh extension) into the DeltaH template whereas what we want to view in this particular instance is the raw data (µcal per injection, designated with a .dh extension). Follow the same procedure as described above to open the layer control dialog box for the DeltaH plot. Move all .ndh files out of the Layer Contents and move the two .dh files from Available Data to Layer Contents, and click on OK. Finally, double-click on the numerical tick labels on the Y axis, which causes the dialog box for the Y axes tick labels to appear. In the factor box in the upper right you will notice a factor of 1000 (which is normally used to convert from cal to kcal). Edit to put a 1 in the factor box, rather than 1000, and click OK. Now the two data sets are plotted in terms of µcal per injection on the Y axis versus a number proportional to injection number on the X axis. You may compare your results with those shown below. The scatter of your points from a straight line should not be much worse than these. If you have already been through the tutorial guide for ITC Data Analysis in Origin, then you may wish to integrate all of your peaks one-by-one, rather than rely on the automatic integration procedure, and this will probably reduce scatter.

-72-


Again, as a gauge of your success you may compare your results to the plot below.

XIII. The Next Step If the data you obtained using 5% sucrose look 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 have not done so. If you are satisfied with your sucrose dilution data, then you might want to move on to real binding experiments. With your instrument, you received a trial kit which includes solutions of the ligand 2'CMP and the enzyme Ribonuclease A. Before your instrument was shipped from MicroCal, we used aliquots of the same samples to generate a binding isotherm and have provided you with a copy of the results we obtained, including the run parameters and the fitting parameters obtained from 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. If everything has gone well to this point, then you should be ready to begin studies on your own samples. Good luck!!

-73-

Section 5.12-Maximizing Baseline Stability

Section 5.12

Maximizing Baseline Stability For demanding experiments where maximum baseline stability is required, it is necessary that the user be aware of small effects which arise from the thermal history of the room in which the instrument is located. It must be recognized that the ITC cell is surrounded by several inches of foam insulation which means that the cell itself is very slow in feeling any effects caused by changes in ambient temperature. For example, the on/off cycles of heating systems and air conditioners, which occur every 10-20 minutes, have almost no effect on baseline position. On the other hand, if the room thermostat is turned down several degrees in the evening and then turned back up at the start of the next working day then the baseline will drift very slowly and will not fully stabilize for many hours even though the room temperature remains constant during the entire working day. Although it will vary from instrument to instrument, the change in equilibrium position of the baseline is of the order of 0.03 µcal/sec for each degree change in ambient temperature. The best way to avoid this problem is to be certain that the room is thermostatted at the same temperature for 24 hours a day when the instrument is being used, if this is possible. It also helps, but does not completely eliminate, this problem if the circulating bath is used continuously (i.e., even when operating near room temperature and above) since this partially shields the cell from changes in room temperature. If most of your experiments are to be carried out at 25 C and above, then you could set the external bath temperature to 20 C and let it circulate continuously. As implied above and pointed out elsewhere, if the temperature of the circulating bath is ever changed, the instrument should be allowed to equilibrate overnight before beginning experiments at the new set temperature.

-74-

Section 5.13-Selecting the Proper Stirring Rate

Section 5.13

Selecting the Proper Stirring Rate The stirring rate on your instrument is user-selectable under Advanced Options in the ITC Setup window, up to a maximum rate of 1500 rpm. In selecting the optimum rate, there is a trade-off between mixing efficiency and baseline noise. Shown in the figure below are

ten-minute baselines at stirring rates from 0 to 925 rpm. It can be seen that the short term noise increases with stirring rate, particularly above 700 rpm, but there is very little change in baseline drift. For almost all your studies, stirring rates of ca. 400500 rpm will give adequate mixing following injections and still provide very high baseline quality. However, for binding studies where the c value (i.e., the product of the binding constant times the concentration of sites in the sample cell) is in excess of 1000, you may obtain better values for the binding constant at stirring rates near 700. The reason for this is that when binding is extremely tight, a significant error is encountered near the equivalence point if the injected ligand solution does not mix completely throughout the entire volume of the sample cell. Finally, when you are studying particulate suspensions which tend to settle from gravity, then more stirring energy is needed to keep a uniform suspension. For phospholipid vesicles which settle slowly, stir rates near 700 rpm should be satisfactory while suspensions of agarose beads which settle quickly will probably require 900 rpm or even higher. About the only way to tell if you are stirring fast enough is to repeat the experiment at a higher stirring rate to see if the shape of the binding isotherm (i.e., heat per injection versus molar ratio of ligand/macromolecule) changes significantly. If it does not, then the lower stirring rate was adequate.

-75-

Section 6-Running a DSC Experiment

Section 6

Running a DSC Experiment Section Contents: 6.1 Using Observer-DSC 6.2 Sample Preparation 6.3 Cell Loading 6.4 Cell Cleaning 6.5 Sideways Operation For Precipitating Proteins 6.6 Troubleshooting 6.7 DSC Experimental Tutorial 6.8 Maximizing Baseline Repeatability

-76-

Section 6.1-Using Observer-DSC

Section 6.1

Using Observer-DSC

Pictured above is the DSC setup window. All DSC experiments are begun by entering run parameters into the appropriate parameter boxes. If your studies require frequent repetition of experiments using the same set of run parameters, then you can use the File: Save File menu option to store a particular set of run parameters to disk. All setup parameters, including selections under Advanced Options, will be stored except the Data Filename and Data File Comments which are specific to each experiment. When you wish to use that same parameter set again in the future, you may recall it through the File: Load File menu option. The DSC Scan Setup window will appear on screen exactly as it was stored, with the two exceptions noted above. This same procedure may be used by calling up an old data file, rather than a Scan Setup file, since Observer will extract all of the appropriate setup information from the header of the data file.

Setting up for Multiple Scans Whenever the user wishes to carry out more than a single scan on the same sample without reloading the sample cell, then the desired Number of Scans would be entered into the appropriate parameter box at the top right of the setup window. Once multiple scans are indicated then the user must make the proper selection in the upper left corner of the setup window before selecting other run parameters. If all run parameters for each scan are to be identical (e.g., upscan followed by second upscan using the same starting and ending temperature), then the All Scans Same option would be selected. If the run parameters for multiple scans are to be different in any way (e.g. upscan followed by downscan) then Unique Scans would be selected by clicking on the appropriate circle to darken it. Run parameters for each run are shown in the file list at the bottom of the setup window (shown above), and the user may scroll through the list to view each individual scan. If the All Scans Same option is active, then any change in run parameters made at the top of the setup window will automatically be entered for each scan in the file listing. If the Unique Scans option is active, then changing run parameters at the top of the setup window will only affect that particular run which has been selected in the file list by first clicking on it. File names for multiple runs are assigned automatically, but may be edited if desired. If the user enters a filename Test, then the first run will be Test1.dsc, the second Test2.dsc, etc. Filenames entered for multiple runs may only be seven

-77-

Section 6.1-Using Observer-DSC characters without the extension (since one extra digit is assigned) and all file names must begin with a letter rather than a digit (e.g., test4 is appropriate, but not 4test).

Defining the Run Parameters Once the Number of Scans has been entered (and the above procedure carried out if there is to be more than one scan) then the remaining run parameters must be assigned in the setup window for each scan. The Starting Temp. which you enter is the temperature where the cells and jacket will equilibrate before the run begins. Once the run starts, there will be a default delay of 5 min (adjustable by user as described later) before data is recorded to allow the baseline to equilibrate. Thus, the actual temperature where data is first available will be slightly different than the starting temperature, depending on scan rate. The Final Temp. is where the experiment will terminate. If you have purchased the downscan option, then a downscan will automatically be carried out whenever the final temperature that you enter is lower than the starting temperature. If you have not purchased the downscan option, then Observer will not accept scans where the final temperature is lower than the starting temperature. Observer will not accept entries for starting or final temperature that are lower than +2 C or higher than 120 C. Entries which you make that are outside of this range will automatically be assigned the appropriate limiting value which is acceptable. These limits have been included to protect your instrument from temperature extremes which might be damaging. If you have reason to go beyond these limits, please contact MicroCal. The Scan Rate may be set from 1 to 120 deg/hr for upscans. It should be recognized however that there is no provision for providing cooling to the jacket and cells during an upscan so the minimum scan rate which can be used for temperatures below room temperature cannot be smaller than the rate at which the calorimeter heats up due to heat gain from the room. Thus, 12 deg/hr is the smallest usable scan rate for upscans starting near 0 C, 5 deg/hr is the minimum if starting at 15 C, etc. For downscans, the scan rate may be set from 1 to 45 deg/hr. In this case, there will be some limitations on the maximum scan rate at low temperatures due to the cooling capacity of the circulating bath used for downscanning. If you wish to downscan all the way to 2 C, then the scan rate you select should be no larger than ca. 30 deg/hr. The Filter Period is the time period over which data is averaged before a data point is stored. For broad transitions such as protein unfolding carried out at a scan rate of 60-100 deg/hr, a filter period of ca. 15-25 sec would normally be appropriate. For very sharp lipid melting transitions, the filter period would have to be much lower than this in order to get a sufficient number of data points on the peak. Of course, the shorter the filter period, the larger will be the short-term-noise. The Resting Temperature is the temperature at which the DSC cell will equilibrate following the last scan in the series. Frequently, you may wish to set the resting temperature to the anticipated starting temperature for the next set of scans to be executed since this will shorten the equilibration time when beginning the next series. In the Cell Conc. box, you should enter the concentration (mM) of the solute which you are studying in the sample cell. When you later call your data file into Origin for data analysis, this concentration will be used to normalize the DSC data on concentration. However, if you should enter an incorrect concentration now, you do have the opportunity to reenter the correct concentration after the data appears in Origin. As indicated above, the Data Filename which you enter may be eight characters (seven if you are doing multiple scans) or less and not begin with a number. You need not enter the extension, as Observer will automatically add .dsc to the filename you enter.

DSC Advanced Options Clicking on the Advanced Options menu item causes the following dialog box to appear. There are several options included here that normally need not concern you on a day-to-day basis but which advanced users may wish to change under certain circumstances.

-78-

Section 6.1-Using Observer-DSC

Delta-T This is the desired temperature difference between the DSC Jacket and it's cells, prior to beginning a scan.

Enable Delta-T Checking With this option selected, the DSC cell will always wait to achieve the specified Delta-T prior to beginning a scan. This ensures that the DSC Cell is well equilibrated prior to scanning.

Enable Battery Usage When a check appears next to this menu option, then battery use is enabled at the MCS for sampling filtered data (run data). When the DSC unit is in a prerun state, both the CFB and JFB preamplifiers are powered by the AC source that the unit is plugged into. Once the prerun is completed and the run begins, if the 'Enable Battery Usage' option is checked then both the CFB and JFB preamplifiers will operate under battery power. The batteries are located in the bottom of the DSC unit, and are constantly being charged whenever the unit is plugged into the AC wall outlet, and the batteries are not in use. By using a battery power for the preamplifiers, you will be minimizing the potential for AC line interference to be seen in the data. Users will always want to use this option, unless there is some problem with the batteries.

Enable Starting Temp. Check When this option is selected, then the starting temperature entered in the Scan Setup window will be achieved before beginning an experiment. If the user is concerned with being at a specific temperature before beginning a run, then this option should be used. In the case where the user is not concerned with the starting -79-

Section 6.1-Using Observer-DSC temperature, then disabling this option will cause the 'Seek Init. Temp.' state to be skipped during the prerun process, and the starting temperature will be the current temperature of the DSC unit.

Desiccation Options Located within the Cell Unit is a small air pump capable of circulating air in a closed loop from the jacket through a small desiccator tube located on the rear of the Cell Unit and back into the jacket. This pump turns on automatically following completion of a scan, and stays on for a period of time controlled by the value entered for Desiccation Period. The purpose of this desiccation step is to remove any moisture which enters into the jacket from very slow exchange with room air. If the Default Desiccation-No thermostatting option is selected, the desiccation begins upon termination of the run at the shutoff temperature and continues for the appropriate time period while the instrument proceeds without interruption to cool the cell in preparation for the next run. If the Desiccation-While Thermostatting option is made active, then the cell is thermostatted at the shutoff temperature during the desiccation period and does not proceed to the next step until the desiccation is completed.

PreScan Wait Once equilibration between jacket and cells has occurred at the starting temperature, then the scan begins. Once scanning starts, it requires a certain amount of time before the baseline has equilibrated. The PreScan Wait period is the time which Observer will wait, subsequent to the start of the scan, before beginning to store data which will be saved in the data file.

Data Options Over a broad temperature range, all DSC instruments exhibit a baseline which has same slope and curvature even when identical solutions are in the sample cell and reference cell. Observer provides the option of Real Time Baseline Subtraction to correct your baselines so they appear reasonably flat in real time data display. We suggest that you operate the instrument routinely with this option checked, and so have made this the default selection. Should you ever wish to see uncorrected data, check the Use Raw CFB Data option. If you wish to view the equation which is subtracted in real time, click on Baseline Subtr. Cons menu at the top of the DSC Advanced Options window. Although there are provisions in this dialog box for subtracting a fourth order polynomial, only the first two terms are being used to determine the standard equation for the correction term CFB' to be subtracted from the raw CFB data during each run, i.e. CFB' (mcal/min) = A0 + A1xT, where T is the temperature in degrees Celsius. Note that each of these two constants are determined at the selected scan rate (deg/hr) by a linear equation. The default constants entered for your instrument were predetermined by MicroCal by analysis of a series of water-water scans at different scan rates and will not normally need to be changed by the user.

Apply To All Scans and Exit Your selections on all these Advanced Options will be applied to the scan selected in the upper left scroll box, if there is more than a single scan selected in the Setup. If you wish different options for each scan, then you would repeat the selection procedure with each scan selected, and then click on OK to leave the dialog box. If you wish all scans to have the same Advanced Options, then you would make those selections and click on Apply To All Scans and Exit. We would again emphasize that users generally need not worry about Advanced Options since the default choices made by Observer are those which are appropriate for almost all experiments.

-80-

Section 6.2-Sample Preparation

Section 6.2

Sample Preparation Reference or sample solutions must be properly equilibrated with dissolved air before being introduced into the cells. The point to be aware of is that the solubility of air in water is higher at low temperature than at high temperature. Solutions or solvents that have been previously stored in the refrigerator for a long time must be degassed before they are entered into the cells. Solutions that have been air-saturated at low temperature and not degassed prior to loading will tend to form air bubbles as they are heated in the cells, and this will produce large aberrations in the baseline. Solutions equilibrated for long times at room temperature will normally not require degassing. A diaphragm pump (ca. 27" Hg vacuum) and plastic vacuum chamber placed atop a magnetic stirrer may be used for degassing without excessive boiling. Normally only 3-4 minutes of vacuum is required to degas if the sample is being stirred but much longer without stirring. If volatile buffers or solutes are being used then solutions should be prepared from degassed or pre-boiled water and stored air-free.

-81-

Section 6.3-Cell Loading

Section 6.3

Cell Loading The degassed sample is drawn slowly (to a volume of 2.5 cc) into the glass filling syringe, after the syringe has been cleaned and thoroughly dried or rinsed. Air bubbles, particularly at the needle end of the syringe, should be avoided. The long needle of the syringe is inserted into the appropriate reservoir of the sample or reference cell and allowed to slide down the narrow stem that connects with the cell. During filling the syringe needle should be a millimeter or so above the bottom of the cell so that liquid flows freely from the needle. The plunger of the syringe is depressed slowly so liquid enters the cell at a uniform rate until enough liquid has been dispensed (ca. l.4 cc) so that the level is just visible in the bottom of the reservoir. The cell itself will be totally filled but there will still be bubbles stuck in the stem because of its narrow diameter. Force the bubbles out of the narrow stem by sharply depressing the syringe plunger to produce a rapid 0.l cc stroke. This should be repeated five to eight times until the liquid level has reached the top of the reservoir. These short strokes should be sharp enough so that liquid spurts up in the reservoir with each stroke. It may require some practice before the technique of delivering short rapid strokes is mastered but this must be done since any bubbles that are still lodged in the lower part of the stem at the time the run begins will produce significant baseline aberrations. If the operator finds it necessary in the beginning, the procedure can be repeated. That is, after the liquid level has reached the top of the reservoir, the syringe plunger can be very slowly raised without removing the syringe from the cell, until the liquid level is again at the bottom of the reservoir. The short strokes can then be repeated until the liquid again comes to the top of the reservoir. After the filling is completed, and the syringe needle is withdrawn, the excess liquid in the reservoir can be removed down to the stem tubing, if desired. MicroCal recommends that you do not dry the cells prior to filling as this is very time consuming. After the final water rinse, your cell should be drained using the long-needle syringe, rinsed twice with your buffer, and finally filled with your sample solution. Due to the small amount of buffer which will still be in the cell after rinsing, your sample will be diluted in concentration by ca. 1.5 - 2% and you can make this correction if it's concentration was determined before it was entered into the cell.

-82-

Section 6.4-Cell Cleaning

Section 6.4

Cell Cleaning In order to obtain satisfactory performance from your instrument, both sample and reference cells must be filled with absolutely no bubbles in the cell or in the filling tube close to the cell. Clean cells are much easier to fill properly because of the tendency of bubbles to form and adhere to foreign matter which might be on the inside surface of the cells. Best results are obtained if the cells are cleaned before each new sample is loaded. When carrying out a series of similar experiments in the same day, it may be satisfactory to drain the sample cell after each experiment, rinse several times with the new buffer, and finally fill with the new sample solution. However, this procedure of rinsing, but not cleaning, the cell between experiments will not be satisfactory if done routinely on a day-to-day basis. We suggest that each new series of experiments begin with a cleaning of the sample cell and reference cell. Harsh detergent solutions (e.g., 20% aqueous solution of Top Job, 5% SDS with 3% dithiothreitol in .02 M Tris pH 8.5) pre-heated to 75 deg. C are usually adequate for cleaning cells that have been exposed to proteins, membranes, or lipids. Neat methanol is also a good lipid solvent. If proteins have heavily precipitated directly in the cell, you may want to precede the detergent cleaning with exposure to a solution containing a low-specificity protease, or to half-concentrated nitric acid at 80 C, to hydrolyze. If using nitric acid, all of the usual safety precautions should be followed strictly (e.g., use safety goggles, protective gloves and apron, draw cold nitric acid into the syringe to load cell-scan to 80 C- incubate- cool instrument- withdraw cold nitric acid, etc.). We have found that for some precipitated proteins, 30 minute incubation with concentrated nitric acid at room temperature is sufficient while for others the high temperature exposure is needed. Always use the 2.5 ml glass syringe with the sealed-in-glass needle when transferring the acid solutions. Never use the vacuum cleaning apparatus with acid solutions except after all the acid has been drained from the cells. The cells are fabricated from Tantalum, which has excellent resistance to most common reagents. However, the DSC cells should not be exposed to concentrated solutions of strong bases, hydrofluoric acid, or hot concentrated sulfuric acid (commonly called "Cleaning Solution") . A brochure has been included that gives more details on Tantalum resistance to common reagents. To use the cell cleaning apparatus which you received, the long needle on the cleaning apparatus is inserted into the filling port of one cell and the apparatus pushed down carefully into the reservoir until the O-ring has sealed.

The end of the lower section of tubing that is attached to the apparatus should be connected to a one liter vacuum flask through the #8 rubber stopper. The sidearm on the vacuum bottle should be connected to the diaphragm pump system, or to house vacuum. The end of the upper piece of tubing is immersed into a 500 mL beaker which contains the hot detergent cleaning solution. The cleaning solution will then be pulled through the cell and into the vacuum flask.

-83-

Section 6.4-Cell Cleaning Once sufficient detergent solution has passed through the cell, the hose is removed from the solution, rinsed free of detergent solution using a plastic wash bottle, and then placed into another beaker containing water for rinsing. After rinsing with ca. 250 cc of water, remove the tube from the rinse beaker and remove the cleaning apparatus from the cell. Remove the remaining water from the cell using the 2.5 ml filling syringe. Also using the syringe, rinse the cell twice with buffer to be used in the next experiment and finally remove all of the buffer from the cell. The cell can then be filled with either buffer (reference cell) or with the experimental solution (sample cell) as described earlier. Although rinsing with buffer is essential, we do not recommend drying cells thoroughly after rinsing and before filling since this process is time-consuming. For the sample cell, there will be some dilution of the test solution due to the small amount of buffer which remains on the walls of the cell. This dilution is small (ca. 1.5-2%) and can be corrected for by multiplying the previously-determined concentration by ca. 0.98 or can be avoided by determining concentration after the sample is removed from the cell. The above cleaning procedure should be applied to both cells.

-84-

Section 6.5-Sideways Operation

Section 6.5

Sideways Operation for Precipitating Proteins The DSC Unit may show baseline aberrations when used to study precipitating solutes. The Unit may be tipped on its side to prevent this problem. The sample and reference cells should be filled in the normal way and the cell port reservoirs opened to the nitrogen or air pressure. Once this is done, the cell assembly should be carefully rotated clockwise through 90 degrees so it is resting on the side of its cabinet (the sample side should be down). The instrument is then equilibrated and run in the normal way while it rests in this sideways configuration. It must then be turned upright again after the run for cleaning and refilling (when filling, the excess liquid should be removed from the cell-port reservoir to the top of the cell stems). The reason this procedure is effective for precipitating solutions has to do with the coin-shaped cell geometry. When the cells are in their upright position, all of the precipitate settles into the narrow bottom of the cell, which has very small surface area. This interferes with the normal convection currents in the sample. In the sideways configuration, the precipitate is distributed over a much larger surface area, which considerably lessens the disturbance of convection patterns. Our experience is that the solvent-solvent or water-water baselines change in the sideways configuration, so that these will have to be run separately and subtracted appropriately from the sample scans run in the sideways configuration. If your laboratory works routinely with proteins which precipitate, the process of sideways operation can be made considerably more convenient using the DSC Rotating Frame accessory from MicroCal.

-85-

Section 6.6-Troubleshooting

Section 6.6

Troubleshooting Whenever you suspect that your DSC instrument is malfunctioning, run a water-water baseline. If your water-water baselines look fine but your sample-buffer baselines seem peculiar to you, then in all likelihood the problem resides in your sample and not in your instrument. Any instrument malfunction should show up in both the water-water and sample-buffer scans. Nearly all non-electronic problems in your instrument which show up in water-water baselines will be associated with one of the following: 1. Cell(s) filled incorrectly. Any bubbles which remain in the cell or in the filling tube close to the cell will lead to bad baselines (bumps, curvature, etc.). Reread the prescribed procedure for filling cells and repeat water-water baselines. 2. Cell(s) not thoroughly cleaned. Dirty cells not only make it more difficult to fill correctly, but can cause problems in the baseline even if the cells are filled correctly. Reread the recommended cleaning procedures, and try a more thorough cleaning procedure than you have used in the past. 3. Moist air in the Jacket. Your jacket is automatically desiccated after completion of each run. However, the desiccation process is only as good as the desiccant which is being used. Examine the Drierite in the desiccant tube on the rear of your Control Unit. If it has begun to lose its blue color, replace with fresh Drierite (or you may purchase replacements for the complete tube desiccator, part # 07 578 5 from Fisher Scientific). After replacing, scan your instrument to ca. 100 C, turn on the desiccator pump (from Observer) for one hour and repeat experiment.

-86-

Section 6.7-DSC Experimental Tutorial

Section 6.7

DSC Experimental Tutorial The following experimental tutorial is designed to acquaint you with the basic features of both the hardware and software of the DSC instrument, as well as to provide experience with several manipulations which must be mastered in order to get the highest quality data from your instrument. Rather than beginning experimentation on the DSC using precious biological samples, we strongly suggest that each user of the DSC instrument complete the following tutorial first, using water in both the sample and reference cells, so that irreplaceable samples are not wasted while mastering the appropriate techniques. At this point, your instrument should have been completely assembled according to the directions provided in the Setup section of help. All electrical connections should have been made, and all MicroCal software installed on the hard drive of your computer (this was done for you if you purchased your host computer from MicroCal). The recommended procedures for preparation of solutions, degassing, filling the cell , etc. have already been discussed in the DSC Experimental Procedures section and the following tutorial assumes your familiarity with those procedures.

I. Sample Preparation Begin by degassing (with stirring) ca. 100 ml of distilled water for ca. 5 minutes using your Gast diaphragm pump (or suitable substitute) and degassing apparatus. This degassed water will be the solution which will go into both the sample cell and reference cell.

II. Filling The Cells The Cell Entry Ports consist of two 3/8" holes, just off center in the white cell port assembly located on top of the DSC Cell Unit. Unscrew and remove the cell port cap to allow access to the Cell Entry Ports. As you face the front of your DSC Cell Unit, the entry tube to the sample cell is on the right and the entry tube to the reference cell is on the left (If overhead lighting is poor, you may want to examine these using a flashlight). Load the 2.5 ml glass filling syringe (8" needle) with the degassed water, and tap the syringe bottom after loading so all bubbles float to the top surface. Insert the long needle of the syringe into the appropriate reservoir of the reference cell (on left side) until you feel the bottom of the cell with the syringe. Depress the plunger of the syringe slowly until the water level is just visible in the bottom of the reservoir. Force any bubbles out of the narrow stem by sharply depressing the syringe plunger to produce a rapid 0.l cc stroke. This should be repeated several times until the liquid level has reached the top of the reservoir. After the filling is completed, and the syringe needle is withdrawn, the excess liquid in the reservoir can be removed down to the stem tubing, if desired. Repeat this procedure for the sample cell. Screw the cap onto the Cell Port tightly, and activate the pressure from the nitrogen tank (or house air) to 15 - 30 psi.

III. Preliminary Operations Using Observer Once your instrument is up and running routinely, we recommend that power to the Control Unit be left on continuously for best performance. For this tutorial however, we want to begin with a power down-power up situation to illustrate a point. Using the AC power switch on the rear of the Control Unit, turn the control unit off and leave it off until prompted by Observer to turn it on. At the host computer terminal, double-click on the MCS Observer icon located in the Windows Program Manager. A large window displaying MicroCal's logo will appear. After opening a serial port for MCS communications, the Observer will try to communicate with the Control Unit. Because the power has been turned off, memory at the on-board computer in the Control Unit has been cleared so its software operating system, MCS OS, is not available to establish contact with Observer at the host computer. When contact cannot be made by Observer, a screen with four user options (and help) will appear.

-87-


Click on the 'Download MCS OS' option. Wait for the prompt to turn on the Control Unit's power. Failure to do so will prevent the file transfer from executing. Once prompted to power up the MCS Control Unit, doing so will initiate the download process. You will see the % complete indicator increasing until all files have been received. After approximately a 20 second delay (while files are decompressed at the Control Unit), Observer will again attempt to get the Cell-Configuration information from the Controller, this time successfully. Observer will configure itself according to the number and types of calorimetric units included in your system, and the main window of Observer will appear. Additionally, Origin will be running in the lower right corner of the screen. Your screen should now appear as pictured below (picture assumes Cell 1 is ITC and Cell 2 is DSC).

-88-


By observing the data channel displays on the main window of Observer, you can verify that data is continuously being received from the Control Unit. If you see the values in the text boxes changing approximately every 5 seconds (though only slightly) then you are ready to continue with this tutorial. Data exchange from the Control Unit to the host can also be verified by observing the red Transmitting LED on the front of the Control Unit. This LED is strobed every time the Control Unit sends data to the host computer. We will start by getting the screen display of the host computer such that we can easily see everything that is going on. First, move the Observer main window to the upper left area of the video screen. Now, under the Options menu, click on Cell Display so that the three submenus appear: Display Cell 1, Display Cell 2 and Display Both Cells. Click on the appropriate one to display only the data from the DSC cell we are working with. If you only have one cell then you may ignore this step. We now should have just one cell's data channels being displayed. Confirm that we have the correct cell displayed by seeing that the cell status text box reads 'Cell Idle-DSC Scan #0'. Users should note that any time only one cell is displayed, by double clicking on the Cell Status text box the display will toggle to the other cell's data. Second, move the Origin window to the upper right most area of the screen. Put the mouse cursor on top of the lower left corner of the main Origin window so that the cursor becomes a double arrow. Now click the left mouse button and while holding the button down, drag the corner of the window so that Origin's main window occupies most of the video screen. Do not make the Origin window so big that you can no longer see the MCS Observer. Now click on the maximize button (upward arrow in the upper right corner) of the appropriate Origin plot window (not the main Origin window) so that it occupies the entire main Origin window. You now should be able to see both the MCS Observer as well as the Origin plot window where your data will be plotted, as shown below.

-89-


III. Setting Run Parameters From the main DSC menu (Cell 1 or Cell 2 menu option) click on the DSC Setup menu option. The DSC setup window will appear. If you are configured for two DSC cells, make sure that you are working with the same cell in both hardware and software.

-90-


In the upper left corner, click on All Scans Same. The button to the left of the All Scans Same text should be blackened indicating that this is the active choice. Now, working from top to bottom in the text boxes, we will define the experimental parameters. -Enter 2 in the Number of Scans text box -Enter 5 in the Starting Temperature text box. -Enter 90 in the Final Temperature text box. -Enter 90 in the Scan Rate text box. -Enter 15 in the Filter Period text box. -Enter 25 in the Resting Temperature text box. -Since we are not actually working with samples for the tutorial, you may leave the Concentration In Cell text box as it is. When working with samples be sure and put the appropriate concentration into this text box, as it will be used during data analysis calculations. -Type dsctest in the Run Data Filename text box. You need not type the filename extension, however regardless of what extension is typed in this text box the datafile extension will be .dsc. Observer will append the scan # to your data filename (dsctest1.dsc) , and the data filename for scan #2 is assigned automatically (dsctest2.dsc). At this point we are set up to do two identical scans. The scans will start at 5 deg., and will scan up to 90 deg., at a rate of 90 deg/hr. The data will be averaged over 15 seconds, resulting in approximately 170 data points. After the last scan is completed, the DSC Cell Unit will go to a Resting Temperature of 25 degrees. Let's change the second scan to be slightly different from the first one. Click on the Unique Scans option in the upper left corner. Now, locate the scan list box at the bottom of the setup window. You can see that there are parameters for two identical scans. Click on scan #2 in the scan summary table located at the bottom of the setup window. By highlighting a scan in the scan summary table, you are making it the active scan for editing. Now enter 100 in the Scanrate text box. You should have seen the scan list change to indicate that scan #2 will have a scanrate of 100 deg/hr. Since we previously selected Unique Scans, only the scanrate for scan #2 is affected. All run parameters for each scan may be changed by following this procedure. For this tutorial, however, we would like to do two identical scans. To restore the run parameters as they were, highlight scan #1 in the scan summary table. Clicking on All Scans Same will assign all run parameters the values for scan #1. The scan summary table should again indicate that the run parameters for scans #1 and #2 are identical. Click on the Advanced Options menu; the DSC Advanced Options window will appear.

-91-


The default values for the DSC Advanced Options will be displayed, as shown above. Users should refer to the DSC Setup topic of help for a detailed description of the options. The options may be changed per individual scan, regardless of the All Scans Same, Unique Scans option in the main DSC Setup window. To change the settings for a particular scan, highlight the desired scan # in the scan list box provided, and make the appropriate changes. To assign the currently displayed options to all scans, click on Apply To All Scans and exit. The displayed options will be assigned for all scans, and you will be returned to the Setup window. For this tutorial we will simply use the default values. Click on OK and you will be back in the DSC Setup window. Now click on the File:Data File Comments menu option. The data file comments window will appear.

-92-

Section 6.7-DSC Experimental Tutorial You may type in any comments that you want to be added to the data file headers. By highlighting the appropriate scan in the scan list box, you may add comments to each scan individually. When you have entered all of your comments, click OK to return to the DSC Setup window. Now click on the File:Save menu option. The Save File window will appear.

Type in dsctest.scn and click on the OK button. You now have saved these DSC run parameters to disk and they are available to you for later use. By default, all setup files like the one that you just saved will reside in the c:\mcs\setup subdirectory. Data File Comments, Cell Concentration and Run Data Filename are not saved to the setup files since they are typically unique to each experiment. All Advanced Options settings are saved to the setup files. To demonstrate the reuse of setup files do the following: Enter 5 in the Number of Scans text box. Now click on All Scans Same. The scan list box at the bottom of the DSC Setup Window displays 5 identical scans. Now click on the Load File submenu. The File:Load window will appear, and all of the DSC setup files (.scn extension) will be listed on the left side.

Click on the dsctest.scn entry that you just created and choose 'OK. The parameters from this file will appear in the DSC Setup Window. Confirm that the Number of Scans text box again displays 2, and that Unique Scans is chosen in the upper left corner. Additionally, the DSC Advanced Options will be as you previously defined them.

IV. Beginning The Experiment Now let's begin the DSC experiment. Click once on the Execute Run button found on the left side of the setup window. A small window will appear on top of the setup window. Follow along with the messages as the run parameters are transmitted to the MCS Control Unit, and the run is started. You should now click on the 'Close' button on the left side of the setup window (or

-93-

Section 6.7-DSC Experimental Tutorial you may minimize the window by clicking on the upper right most down-arrow of the window). The DSC setup window will no longer be visible (or will be minimized). It should be realized that windows are best to be closed when you are finished using them, rather than simply minimized. Open windows exhaust system resources, and thus should be closed when no longer needed. If you need to view the Setup window later you may recall it using the Cell 1(or 2):DSC Setup menu option. By looking at the Cell Status box of the main window (bottom most text box), you can follow along with DSC cell as it progresses through several prerun states. It should now be in the Seek Init. Temp. state, with the temperature linearly decreasing towards 5 degrees.

V. Manipulating Origin If you look at the appropriate plot window in Origin, you can watch the prerun data being plotted versus time. The legend at the top of the plot window indicates the colors of the different data channels being plotted. The blue dataset in layer 1 is the Cell Feedback, the red dataset in layer 2 is the Delta T, the black dataset in layer 3 is the jacket temperature and the green dataset in layer 3 is the bath temperature. The units of each dataset can be found directly above their respective axis.

You can see that there are three different Y-axes in this plot window. The Jacket Temperature (black dataset-layer 3) and the Bath Temperature (green dataset-layer 3) are now both decreasing towards the starting temperature for scan #1 (5 degrees). Click once on the ON/OFF button located in the lower right corner of the Origin plot window. Two other buttons will appear, as well as slide bars on each of the Y axes. On the rightmost Y axis (layer 3), click and drag the bottom slide bar to 0 and the upper slide bar to 10. Now click on the Zoom button located in the bottom right region of the plot window. The Jacket/Bath Temperatures should now be displayed on a scale from 0 to 10 (don't worry if your data has gone off scale). You may zoom in on any layer by following these steps in the appropriate layer. The legend at the top of the plot window, along with the axis label colors indicate which dataset is plotted in which layer. To zoom out on a plotted layer, move the slide bars beyond the Y axis minimum and maximum values for the appropriate layer (we are working in layer 3), as shown below.

-94-


Now by clicking on the 'Zoom' button, layer 3 will zoom out. The zoom out factor is found behind the 'Values' button. We will discuss the 'Values' button immediately below. As an alternative to using the slide bars/Zoom button sequence for changing the display range, let's zoom back in on layer 3 using another method. Double-click on the Y axis line for layer 3, which brings the Y Axis Dialog Box into view. In the upper left corner of this dialog box, you will see Y axis limit boxes designated as From: and To: The From: box is darkened and ready to edit. Enter 0 in the From: box. Press the tab key on your keyboard, which then darkens the To: box for editing. Enter 10 in this box. Click on OK and the dialog box closes. The temperature datasets will now be displayed on a full scale of 10 degrees. You may scale any of the Y axes displays using either of the these two methods. The X axis has no zoom feature. Demonstration of Auto Scroll/Scale Feature: Click once on the Origin control labeled 'Values' in the lower right area of the plot window. A simple dialog box will open.

-95-


The first entry in this dialog box is the zoom out scale factor. As you can see, the factor is set to 2, which means that a zoom out will result in twice the Y axis range, centered about the same point as is the current scale. You may set this value directly from this text box. The checkbox labeled 'DDE Scale or Scroll?' allows you to turn on/off the auto scroll/scale feature. When the box is checked then the feature is active for the X axis. By clicking on the 'Which?' listbox, you may change the selection to either Scale or Scroll. When Scale is selected (and active), whenever the displayed data exceeds the current X Axis display, then the X Axis will be scaled by the factor appearing in the 'Fraction of Full Scale?' text box. Likewise, when Scroll is selected (and active), whenever the displayed data exceeds the current X Axis display, then the X Axis will scroll by the factor appearing in the 'Fraction of Full Scale?' text box. Here, the total full scale range will remain the same, but will shift to a different window of values. Finally, the 'First DDE on Screen' check box indicates whether or not the data display will be scaled or scrolled relative to the first data point that is received. Users will probably want this option left as active. When the jacket temperature reaches 5 degrees (black dataset in layer 3), the DSC cell will exit the Seek Init Temp state and will enter the Check Delta T state. When the Delta-T signal (red dataset in layer 2) becomes less than the specified Delta T for the scan (we asked for a Delta T of .1 deg. in the Advanced Options window), the DSC Cell will go into the Prescan Wait period.

VI. Baseline Equilibration The DSC Cell should now be in the Prescan Wait state as indicated in the Cell Status box of the appropriate cell. Upon entering this state the heaters of the DSC cell are turned on, and the actual scanning of the cell is underway. The sudden blast of heat causes a disruption in the cell's equilibration, and the purpose of this state is to allow for reequilibration of the cell and jacket, prior to generating and saving data for the scan. When the Prescan Wait period has elapsed, the state will be exited, and your cell will enter the Scanning state. The default value for Prescan-Wait in the Advanced Options window was 5 minutes.

VII. Data Collection The DSC cell is now scanning, and in the Scanning state. Within a short time, a new Origin plot window will appear, and all ensuing experimental data will be plotted in it. Initially, the CFB data will be displayed on a full scale of 10 mcal/min, with your first data point centered within that range. If users decide after beginning a scan or set of scans that they would like change any of the Run parameters, then simply make the changes in the DSC setup window, and click on Execute Run. The new run parameters will be transmitted to the Control Unit, and the scans will continue using these new run parameters. Of course, changes to parameters for scans which are completed will have no effect.

VIII. Entering a Calibration Pulse Allow the cell to scan for ca. 25 degrees. By the time the temperature data channel reads ca. 30, we should have a significant portion of the baseline established. We are ready to enter a calibration pulse for scan #1. From the main DSC menu (Cell 1 or Cell 2 in Observer), click on the Calibrate DSC submenu. The DSC Calibration window will appear. It is here that you will enter a calibration pulse size, as well as turn the pulse on, and eventually off.

-96-

Section 6.7-DSC Experimental Tutorial Edit the Pulse Size entry to read 5 mcal/min. Click the Pulse On button to administer the calibration heat pulse. Once the calibration pulse has been turned on, you will not be able to close the DSC Calibration window until the pulse has been turned off. For now, it's a good idea to minimize the window (click on the downward arrow in the upper right corner) so that we do not lose sight of it behind another larger window. If this should happen, when you again want access to the DSC Calibration window, simply select it again from the main DSC menu (Cell 1 or Cell 2 in Observer). The window will again appear. To view your data, click on the Origin window to make it active. The cell feedback (blue dataset in layer 1) should be rapidly increasing until it eventually levels off and establishes a new deflected baseline. In a well calibrated DSC Cell, the baseline deflection is the size of the pulse we applied to the cell (i.e. 5 mcal/min). Leave the pulse turned on for 5 minutes so that we establish a significant amount of the deflected baseline after it flattens out. Finally, turn the pulse off by clicking on the Pulse Off button. Click on Close and the DSC Calibration window will disappear. The Cell Feedback (CFB) data will decrease sharply until it levels off at the original baseline. After both scans are completed (do not enter a calibration pulse on the second scan), the data files can be found on your hard drive in the \MCS\DATA subdirectory.

IX. Plotting and Analyzing Data In Origin At this point you should have completed two DSC scans. We can now go to Origin to analyze the data. Click on the Origin-DSC Data Analysis icon in Program Manager. You should see the DSC raw data template as pictured below.

If this is not what you see, then click once on the Origin 'Edit' menu, and then again on the 'Change Menu'\'ITC Data Analysis' submenu. You may answer yes if prompted to start a new session. You now will be looking at the Raw DSC data template. Your raw data from the DSC are in units of mcal/min. Normally when you read raw data files into Origin, they will be automatically normalized on scan rate and divided by 1000 to convert from mcal/min to cal/deg. The only exception to this is when you have entered a calibration pulse to check that the Y-Axis calibration constant is correct. That is the present goal in this tutorial. You will see a button to the right of the Origin graph which says Scan Rate Normalization. Click on the check mark to remove it, so the data will not be normalized on scan rate and devided by 1000. To import your MCS DSC data into the plot window, click on the MCS Data button. The File Open dialog box will open. Find your way to the \MCS\DATA subdirectory, select the dsctest1.dsc entry and click 'OK'. Origin will automatically plot the raw data. Follow the same procedure to import the dsctest2.dsc data file. You now have the raw data from both scans plotted in Origin.

-97-


Pictured above is the raw data obtained from the same sets of scans as in this tutorial, generated at MicroCal. Though each DSC Cell has a unique characteristic baseline, you can use this data as a gauge of how good your own experimental data is. Typically, the slope and curvature of the DSC baseline will vary from cell to cell, but the repeatability of your scans should be comparable to the sample data pictured. For more details on Baseline Repeatability, read Section 6.8. In order to verify that your instrument is calibrated, as well as to get some exposure to some Origin data analysis techniques, we will perform a reference data subtraction using the data from the two scans you've just executed. From Origin, click on the Subtract Reference...button located on the RawDSC window. A dialog box will appear. Click on the DSCTEST1.DSC_CP dataset from the list of available datasets to select it. Now click on the upper most arrow (==>) to move the dataset into the Y1: text box. Repeat this procedure to move the DSCTEST2.DSC_CP dataset into the Y2: text box. You can see that the middle text box defines the operation to perform on Y1: and Y2:, in this case subtraction. The resulting dataset will still be DSCTEST1.DSC_CP as indicated next to the Y: at the top of the dialog box. Your dialog box should appear as shown below.

To subtract the dsctest2.dsc (no calibration pulse) data from the dsctest1.dsc data (5 mcal/min pulse applied during scan), click on the OK button and the operation will be carried out. You will be left with the resulting dataset displayed, as shown below.

-98-


To verify that your instrument is well calibrated, click on the data reader tool in the Origin Toolbox. Locate this in the baseline immediately prior to having turned on the calibration pulse and click once. The Y-Axis value for this data point will appear in the RAWDSC status bar at the top of the window. This should be very close to zero. Then locate the data reader tool in the flat portion of the baseline at the top of the calibration pulse and click once. The Y-Axis reading here should be very close to 5 mcal/min, i.e. the value of the pulse entered during the run.

XI. The Next Step If the data you obtained for the two water-water scans look 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 have not done so. If everything has gone well to this point, then you should be ready to begin studies on your own samples. Good luck!!

-99-

Section 6.8-Maximizing Baseline Repeatability

Section 6.8

Maximizing Baseline Repeatability For demanding experiments where the maximum degree of baseline repeatability is required, the user should be aware of small effects which arise from thermal history of the DSC cell unit. If you begin an experiment after the cell has been resting overnight at room temperature, the baseline will be slightly different than if you begin an experiment immediately after completing an earlier experiment which ended at 100 C, for example. The reason this occurs is due to the very slow thermal equilibration of the foam insulation which surrounds the cell. For maximum accuracy, you should try to minimize the effects from thermal history which occur before the buffer-buffer scan and the sample-buffer scan. The best way to do this is to carry out a "dummy" scan as the first run of the day, before doing a series of real scans, using the same run parameters. A second way is to run two buffer-buffer baselines (i.e., one after the cell has rested overnight at room temperature and one after the cell has cycled to high temperature) and to use the appropriate baseline for subtraction which has the same thermal history as the sample-buffer run.

-100-

Section 7-Calibrating the Cells

Section 7

Calibrating the Cells Section Contents: 7.1 ITC Cell Calibration 7.2 DSC Cell Calibration

-101-

Section 7.1-ITC Cell Calibration

Section 7.1

ITC Cell Calibration Section 4.2 describes the ITC Calibration Setup window in detail, and this section assumes your familiarity with it. All ITC cells have been calibrated at MicroCal, prior to shipment. It is good practice to verify the calibration of your cell every 3 months by following the procedures discussed in this section. When setting up an ITC calibration experiment, it is best to enter calibration heat Pulse Sizes on the order of 10 µcal/sec or larger, in order to negate any effects from slight baseline drift. Additionally, users must enter Pulse Durations on the order of several minutes. This allows the cell feedback to reequilibrate itself and to establish a deflected baseline. The size of this baseline deflection is the determining factor for verifying calibration. Finally, users will want to allow enough time before the heat pulse is turned on to establish the baseline, and after the heat pulse has been turned off to allow the cell feedback to return to it's original baseline. Entering an initial delay of at least one minute will allow the baseline to be established prior to administering the heat pulse. Entering a value for Time Between Pulses which is 4 minutes longer then the Pulse Duration will allow the deflected baseline to return to it's original value.

Calibrating to Baseline Deflection: Once the ITC calibration data has been generated and the run completed, inspection of your data will verify calibration of your ITC cell. Users may find it sufficient to verify calibration from within the MCSDATA.ORG document (document used to display all MCS data via DDE) using the data reader tool. To do this, simply click on the data reader tool from Origin's toolbox. If you do not see the toolbox in Origin select the Options:Toolbox menu and it will appear in the upper right corner of the document. The data reader tool is shown below.

After selecting the data reader tool, click on the CFB dataset in the Origin plot window. Origin will pick the closest data point to where you have clicked and display the X and Y coordinates of that point in the status bar at the top of the window. You may now move through the dataset point by point using the left and right arrow keys on the keyboard. Move the data reader along the CFB trace until you are at a point that well represents the position of the original baseline (before heat pulse was turned on). Record the Y-Axis value at that point. Now move the data reader to a point that well represents the position of the deflected baseline (after CFB has responded and reequilibrated during heat pulse) and record the Y-Axis value. Subtracting the first Y-Axis value from the second will yield the magnitude of the baseline deflection. The result should agree with the pulse size that was entered to within one percent. If the difference is greater than one percent then you may adjust the Y-Axis calibration constant by multiplying the current constant by the ratio of the expected deflection to the actual deflection in the baseline, i.e. New Constant = Old Constant x (expected deflection/actual deflection). Make the appropriate changes to the Y-Axis calibration constant via the Calibration Constants window, and save your changes. Try the calibration experiment again to assure yourself that the new constant is satisfactory.

Calibrating to Area Under Heat Pulse: -102-

Section 7.1-ITC Cell Calibration

As an alternative to using baseline deflection for verifying calibration of your ITC cell, users may prefer to compare the area under the heat pulse to that of the expected area. Again, users should follow the guidelines stated above when entering values for the Pulse Size and Pulse Duration in the calibration setup window. Once the calibration data has been generated and the run completed, you may read the datafile into Origin as you would an ITC titration datafile (See Section 6.7 for brief description, ITC Data Analysis in Origin Manual for detailed description). To analyze your ITC data, click on the Origin-ITC Data Analysis icon from Program Manager. Origin will open, and the RAWITC template will be displayed. Once you have the calibration data read into Origin, there are two things to you will need to keep in mind: 1.) You need to plot the unnormalized area (*_dh dataset as opposed to *_ndh dataset) in the Origin Delta H plot window when comparing results. 2.) You will need to change the X-Axis from Concentration of ligand to Pulse number. By comparing the integrated areas to the expected areas we can confirm calibration of your ITC cell. You can read the integrated area from the Delta H plot window (use data reader tool), or from the Origin worksheet, once you have plotted the unnormalized (*_dh dataset) area data versus pulse number. To compute the expected area simply multiply the Pulse Size by the Pulse Duration i.e. (µcal/sec) x (sec) = µcal The result should agree with the integrated area to within one percent. If the difference is greater than one percent then you may adjust the Y-Axis calibration constant by multiplying the current constant by the ratio of the expected area to the actual area, i.e. New Constant = Old Constant x (expected area/actual area). Make the appropriate changes to the Y-Axis calibration constant via the Calibration Constants window, and save your changes. Try the calibration experiment again to assure yourself that the new constant is satisfactory.

-103-

Section 7.2-DSC Cell Calibration

Section 7.2

DSC Cell Calibration All ITC cells have been calibrated at MicroCal, prior to shipment. It is good practice to verify the calibration of your cell every 3 months by following the procedures discussed in this section. When doing a DSC calibration, it is always best to carry out two scans, both of which are identical in all respects (run parameters, thermal history, etc.) other than the heat pulse itself. Once the DSC calibration data has been generated, inspection of your data will verify calibration of the DSC cell. To analyze your DSC data, click on the Origin-DSC Data Analysis icon from Program Manager. Origin will open, and the RAWDSC template will be displayed. Your raw data from the DSC are in units of mcal/min. Normally when you read raw data files into Origin, they will automatically be normalized on scan rate and devided by 1000 to convert from mcal/min to cal/deg. The only exception to this is when you have entered a calibration pulse to check that the Y-Axis calibration is correct. You will see a button to the right of the Origin graph which says 'Scan Rate Normalization'. Click on the check mark to remove it, so the data will not be normalized on scan rate and devided by 1000. To import your DSC data into the plot window, click on the 'MCS Data' button. The File Open dialog box will open. Find your way to the \MCS\DATA subdirectory, select one of the data files you have just generated and click 'OK'. Origin will automatically plot the raw data. Follow the same procedure to read in the second data file. You now have the raw data from both scans plotted in Origin. In order to verify that your instrument is calibrated we will perform a reference data subtraction using the data from the two scans you've just executed. From Origin, click on the 'Subtract Reference...' button located on the RAWDSC window. A dialog box will appear. Click on the dataset which contains the calibration heat pulse from the list of available datasets to select it. Now click on the upper most arrow (=>) to move the dataset into the Y1: textbox. Repeat this procedure to move the second dataset (no heat pulse applied) into the Y2: text box. You can see that the middle smaller text box defines the operation to perform on Y1: and Y2:, in this case subtraction. The resulting dataset will retain the name of the Y1: dataset, as indicated next to the Y: at the top of the dialog box. Your dialog box should appear as shown below, although the actual dataset names may differ.

To subtract the second dataset from the first, click on the 'OK' button and the operation will be carried out. You will be left with the resulting dataset displayed. Since the characteristic baseline will now be very nearly zero, you may simply read the baseline deflection using the data reader tool from the Origin toolbox. If you do not see the Origin toolbox, select the Options:Toolbox menu and it will appear in the upper right corner of the Origin document. The data reader tool is shown below.

-104-

Section 7.2-DSC Cell Calibration

After selecting the data reader, click on the CFB dataset in the Origin plot window. Origin will pick the closest point to where you have clicked and display the X and Y coordinates of that point in the status bar at the top of the window. You may now move through the dataset point by point using the left and right arrow keys on the keyboard. Move the data reader along the CFB trace until you are at a point that well represents the position of the original baseline (before heat pulse was turned on). Record the Y-Axis value at that point. Now move the data reader to a point that well represents the position of the deflected baseline (after CFB has responded and reequilibrated during heat pulse) and record the Y-Axis value. Subtracting the first Y-Axis value from the second will yield the magnitude of the baseline deflection. The result should agree with the pulse size that was entered to within one percent. If the difference is greater than one percent then you may adjust the Y-Axis calibration constant by multiplying the current constant by the ratio of the expected deflection to the actual deflection in the baseline, i.e. New Constant = Old Constant x (expected deflection/actual deflection). Make the appropriate changes to the Y-Axis calibration constant via the Calibration Constants window, and save your changes. Try the calibration experiment again to assure yourself that the new constant is satisfactory.

-105-

Section 8-Customer Support

Section 8

MCS Remote Control Customer Support Each MCS system includes a high speed modem and the remote control software Carbon Copy for Windows. When a telephone line is connected to the modem MicroCal has the ability to call into the customer's host PC. We then see everything on our PC that the customer sees on their PC. We can operate your instrument remotely, exchange computer files, and pull up a chat window so that we can communicate directly on screen during the session. MicroCal welcomes requests for remotely operated tutorial sessions. Please give 48 hours notice on these requests. Prepare for a remote session by connecting the modem to the serial port that the mouse is normally connected to and running the remote control application called Host. The modem will be initialized and an icon will appear labeled Waiting for Call. Then call or fax MicroCal with the telephone number for the line connected to the modem and we will initiate the session.

-106-

Appendix A-ITC Cell Status Definitions

Appendix A

ITC Cell Status Definitions Thermostatting The ITC cell enters this state when a run is terminated or completed. It idles in this state, attempting to maintain its temperature at the currently-set Thermostat Temperature (may be changed in the Set Thermostat Temperature dialog box found within the main ITC Cell 1(or Cell 2) menu). The thermostat temperature can be achieved only if it is higher than effective ambient temperature.

Initializing Injector This state occurs immediately after Execute Run is selected, and the injector system seeks its home position (maximum upward position, swung 90o away from the cell access tubes) whereupon the cell proceeds to Seek Initial Temperature state. If the home position cannot be found, the cell returns to the Thermostatting state.

Seek Init. Temp The ITC cell seeks the experimental temperature that was selected by the user. Once that temperature is reached, the cell passes to the Equilibrating state. Prerun data is displayed in Origin during this state. If the selected temperature is below effective ambient temperature, the cell may stall in this state since there is no way to cool the cell and jacket directly. To avoid this problem, the circulating bath should be used to achieve a lower effective ambient temperature and, after equilibrating for 510 hours after circulation is started, the run may be repeated.

Equilibrating After reaching the experimental temperature, the Control Unit will algorithmically strobe heaters in an effort to establish nearly zero temperature difference between the jacket and the two cells. Once this happens, Observer monitors the CFB until it becomes positive and non-saturated whereupon this state is exited and the instrument is ready for the syringe to be inserted.

Ready For Syringe A button will appear at the bottom of the main window directly below the Cell Status text box indicating that the user may insert the syringe into the sample cell. Once inserted, the user may click on the button labeled Syringe Inserted and the state is exited.

Seeking Syringe Upon receiving the signal that the syringe has been inserted, the injector system algorithmically finds and securely clamps onto the expanded top of the plunger of the injection syringe. A message is sent to Observer indicating the volume of liquid in the syringe which is available for injection, the stirrer is turned on, and the state is exited.

-107-

Appendix A-ITC Cell Status Definitions

Checking Baseline This state allows viewing of the CFB baseline in the Origin plot window prior to beginning a series of injections. When the baseline is sufficiently flat, the user may click on the Baseline OK button below the Cell Status text box whereupon all injections will be carried out automatically according to the directions previously entered in the Setup window.

Making Injection # and Collecting Injection # Once the baseline is OK'd, a new Origin template appears for plotting all CFB data generated during injections. The Cell Status window will keep you informed as to which injection is in progress and Observer will continuously update the available Volume in Syringe if you care to view it in the Setup window. If any errors occur, these will be displayed in the Cell Status window.

Waiting To Resume This state is merely a wait state used during data retrieval. If during data retrieval from an ITC cell the injection schedule is carried out in full, then the cell will wait in this state until the data for the run has been retrieved in full. Once retrieved in full, the cell will then proceed to the Resetting Injector state.

Resetting Injector The injector returns to it's home position. The occurrence of any errors while returning home will be reported to Observer.

MCS Error Indicates that an error has occurred during one of the other states. The errors which may occur concern the temperature of the ITC Cell and the temperature of the external water bath. The minimum and maximum allowable temperatures for both the cell and the water bath are in the calibration constants file. Any time these allowable temperature ranges are violated, then the ITC cell will immediately enter this state, and the bath will be set to 25 degrees Celsius. When the allowable temperature ranges are no longer violated, choose 'Clear Error' from the main ITC menu or from the ITC Setup window to reset the cell, and it will enter the 'Thermostatting' state.

-108-

Appendix B-ITC Calibration Constants

Appendix B

ITC Calibration Constants The ITC calibration constants window is pictured. Definitions of the individual constants can be found below.

Y Axis Constant used to map CFB voltage to µCal/sec.

MCS Temp. Slope/Offset Constants used by MCS Control Unit to map temperature circuit voltage to deg. Celsius.

Bath In Slope/Offset Constants used when setting the bath to a target temperature. Maps selected temperature to voltage for driving the external water bath.

Bath Out Slope/Offset Constants used when reading a bath Temperature. Maps voltage out of bath to temperature reading which is displayed.

-109-


CAL DAC Slope/Offset Constants used to map the Calibration heater power to µCal./sec.

R-HTR Resistance value for calibration heater located on reference cell.

R-SENSE Resistance value for total calibration heater circuit.

Cell Volume The calorimetric volume of the sample cell which is used in all data analysis calculations. This is not the total volume required to fill the cell for obtaining experimental data due to the volume in the cell's access tube.

Jacket Min. T The minimum temperature that an ITC Calorimetric Unit will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging a Calorimetric Unit by freezing it's contents.

Jacket Max. T The maximum temperature that a ITC Calorimetric Unit will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging a Calorimetric Unit by overheating.

Bath Min. T The minimum temperature that an external water bath will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging an external water bath or a Calorimetric Unit by freezing it's contents.

Bath Max. T The maximum temperature that an external water bath will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging an external water bath or a Calorimetric Unit by overheating.

MCS Temp. Polynomial Constants used by MCS Observer to map temperature circuit voltage to deg. Celsius. Function may use up to a fourth order polynomial, and coefficients are listed as A0, A1, A2, A3, A4. By default, only linear mapping is used, and the two constants should be identical to MCS Temp. Slope/Offset above.

-110-


Syringe Constants There are ten lines reserved for syringe constants which convert linear displacement in a syringe to volume of injectant. MicroCal, Inc. provides users with three different size syringes (50, 100, and 250 µl), and their syringe constants (the remaining seven lines are set to 0). Syringe constants can be changed via the Calibration Constants menu option in the MCS Observer.

Stir Speed Constant (Not available from any constants window) Used to map stir speed setting.

-111-

Appendix C-DSC Cell Status Definitions

Appendix C

DSC Cell Status Definitions DSC Cell Idle The DSC Cell enters this state when a run or set of runs is terminated or completed. There is no active hardware control in this state besides the charging of the preamplifiers' batteries.

Seek Init. Temp The DSC cell seeks the starting temperature of the current scan which was selected by the user Once that temperature is reached, the cell passes to the Check Delta T state. Prerun data is displayed in Origin in this state. There is a DSC Advanced Option which allows the user to bypass this state, and to begin the scan from the current temperature (doesn't affect downscans).

Check Delta-T The purpose of this state is to wait until a specific temperature difference between the jacket and the cells (both reference and sample cell) is achieved. In the case of a downscan the delta-T is determined by Observer with a linear function of scanrate. In an upscan the delta-T is determined by the user (in Advanced Options). There is a DSC Advanced Option which allows the user to bypass this state, and to begin the scan with the current Delta-T (doesn't affect downscans, however).

Pre-Scan Wait The purpose of this state is to allow the instrument to completely equilibrate prior to generating experimental data. Heaters are turned on (in a downscan the bath begins ramping down in temperature), and preamplifiers (both CFB and JFB) are under battery power. In the case of an upscan the water bath is no longer needed for the current scan (may have been used to 'Seek Init. Temp'), so it is set to the starting temperature for the next scan, or to the Resting Temperature if it is the last scan. The amount of time to wait in this state for a flat baseline is chosen in the Advanced Options window.

Scanning-Scan # The cell is scanning and the MCS is generating experimental data per the specified filter period. When the first stream of data from this state is received by the MCS Observer (assuming Data Display in Origin is turned on for the cell), then the Origin plot window template for displaying DSC run data is loaded, and all ensuing DSC run data will be displayed in the template.

Desiccating During this state the desiccant system will be circulating dry air through the jacket in an attempt to remove any moisture from inside the adiabatic jacket. The user has access to the duration of this desiccation state via the DSC Advanced Options. The user has control of the way in which the desiccation takes place. Desiccation can take place while the cell is thermostatting or while the cell is cooling. By running the desiccator after an upscan, while thermostatting (the cell will be at the final temperature for the scan just completed), then users will be circulating hot air through the jacket. This is the most effective means of removing moisture from the internals of the jacket.

-112-

Appendix C-DSC Cell Status Definitions

DSC Scan Over This state is merely a check point to see if there are more scans to be carried out. If there are scans remaining then the cell will proceed to the Seek Init. Temp. state of the next scan. If there are no more scans remaining then the cell will proceed to the All Scans Complete state.

All Scans Complete This is the state that a DSC cell will go into when the last scan, of a set of scans, has been completed. When entering this state the bath will be at the user specified Resting Temperature and the bath valves will be opened. By setting the Resting Temperature to the desired starting temperature for the next scan time will be saved during the prerun process of the next scan.

MCS Error This state is used to indicate an error has occurred during one of the above mentioned states. The errors which may occur concern the temperature of the DSC Cell and the temperature of the external water bath. The minimum and maximum allowable temperatures for both the cell and the water bath are in the calibration constants file. Any time these allowable temperature ranges are violated, then the DSC cell will immediately enter this state, and the bath will be set to 25 degrees Celsius. The bath valves will be closed and the DSC cell will be allowed to drift toward room temperature. When the allowable temperature ranges are no longer violated, click on 'Clear Error' to reset the error condition and cause the cell to enter the 'Cell Idle' state

-113-

Appendix D-DSC Calibration Constants

Appendix D

DSC Calibration Constants The DSC calibration constants window is pictured. Definitions of the individual constants can be found below.

Y Axis Constant used to map CFB voltage to mCal/min.

MCS Temp. Offset/Slope Constants used by MCS Control Unit to map temperature circuit voltage to deg. Celsius.

Bath In Offset/Slope Constants used when setting the bath to a target temperature. Maps selected temperature to voltage for driving the external water bath.

Bath Out Offset/Slope Constants used when reading a bath Temperature. Maps voltage out of bath to temperature reading which is displayed.

-114-


CAL DAC Offset/Slope Constants used to map the Calibration heater power to the mCal./min.

R-HTR Resistance value for calibration heater located on reference cell.

R-SENSE Resistance value for total calibration heater circuit.

Cell Volume The effective working volume of the sample cell, which is used in all data analysis calculations. This is smaller than the total volume required to fill the cell for obtaining experimental data due to the volume in the cell's access tube..

Jacket Min. T The minimum temperature that a DSC Cell will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging a Cell

-115-


Jacket Max. T The maximum temperature that a DSC Calorimetric Unit will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging a Calorimetric Unit by overheating.

Bath Min. T The minimum temperature that an external water bath will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging an external water bath or a Calorimetric Unit by freezing it's contents.

Bath Max. T The maximum temperature that an external water bath will be allowed to reach. MCS Control Unit will use this value as a temperature shutoff. This is a safety feature to prevent damaging an external water bath or a Calorimetric Unit by overheating.

Temp. Polynomial Constants used by Observer to map temperature circuit voltage to deg. Celsius. Function may use up to a fourth order polynomial, and coefficients are listed as A0, A1, A2, A3, A4. By default, only linear mapping is used, and the two constants should be identical to MCS Temp. Slope/Offset above

-116-

Appendix D-DSC Calibration Constants The DSC Advanced calibration constants window is pictured. Definitions of the individual constants can be found below.

Cell Main Heater Setting For Low Scanrates Constants used to determine the cell main heater setting for scanrates between 0 and 3 degrees per hour.

Calibration Heater Setting For Low Scanrates Constants used to determine the calibration heater setting for scanrates between 0 and 3 degrees per hour.

Jacket Main Heater Setting For Low Scanrates Constants used to determine the jacket main heater setting for scanrates between 0 and 30 degrees per hour.

Cell Main Heater Setting For High Scanrates Constants used to determine the cell main heater setting for scanrates between 3 and 120 degrees per hour.

Calibration Heater Setting For High Scanrates Constants used to determine the calibration heater setting for scanrates between 3 and 120 degrees per hour.

-117-


Jacket Main Heater Setting For High Scanrates Constants used to determine the jacket main heater setting for scanrates between 30 and 120 degrees per hour.

Seek T1 Heater Settings When a calorimetric unit is in the Seek Init. Temp. state these heater values will be used in order to raise the temperature of the jacket/cells. These heater values (in Hexadecimal range 000=no power to FFF=full power) create the Seek Init. Temp. scanrate.

Downscan Delta-T Offset/Slope Linear mapping used to determine the delta-T setting for a DSC downscan. Function maps downscan scanrate to delta-T in degrees Celsius.

-118-