1. ME4331 – DSC Review PART 2 MAY 1, 2008 Jeung Hwan Choi

2. Prenucleation procedure FREEZE-THAW METHOD Prenucleated cooling and reheating (1xPBS + 2M glycerol) Reference : Höhne et al. Differential Scanning Calorimetry 1996 Perkin Elmer, Inc. supercooling (cooling / heating rate = 5 °C/min)  Sample is cooled to bring about supercooled nucleation  Sample is reheated close to the melting point, allowing small ice crystals to be present  Sample is cooled from a prenucleated state, minimizing non-equilibrium effects

3. Prenuc vs non-prenuc – DSC FREEZE-THAW METHOD Prenucleated vs. non-prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min) prenucleatednon-prenucleated

4. ADD: Prenuc PBS6MG movie FREEZE-THAW METHOD Prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

5. ADD: Prenuc PBS6MG movie FREEZE-THAW METHOD Prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

6. ADD: Prenuc PBS6MG movie FREEZE-THAW METHOD Prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

7. ADD: Prenuc PBS6MG movie FREEZE-THAW METHOD Prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

8. ADD: Prenuc PBS6MG movie FREEZE-THAW METHOD Prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

9. ADD: Prenuc PBS6MG movie FREEZE-THAW METHOD Prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

10. ADD: Supercool PBS6MG movie FREEZE-THAW METHOD Supercooled freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

11. ADD: Supercool PBS6MG movie FREEZE-THAW METHOD Supercooled freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

12. ADD: Supercool PBS6MG movie FREEZE-THAW METHOD Supercooled freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

13. ADD: Supercool PBS6MG movie FREEZE-THAW METHOD Supercooled freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

14. ADD: Supercool PBS6MG movie FREEZE-THAW METHOD Supercooled freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

15. ADD: Supercool PBS6MG movie FREEZE-THAW METHOD Supercooled freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) Reference : Choi & Bischof In Review 2006 (cooling / heating rate = 5 °C/min)

16. DSC outline – latent heat DSC – SOP Preparing the DSC for Operation 1. Turn ON the communication computer and the DSC 2. Turn ON the cryofill by pressing the switch on the cryofill control box (Note) Powered ON standby : The entire system is usually kept ON even during standby. However, you should be aware that the sample chamber may reach temperatures ~ 40 o C during non-operation.

17. DSC outline – latent heat DSC – SOP Checking the DSC Firmware Version 1. From the Windows desktop select : Start -> Programs -> Pyris Software for Windows -> Pyris Configuration 2. Comunication should be with COM2 and the ‘Analyzers’ column should display the Pyris 1 DSC. From this screen, press the Edit button.

18. DSC outline – latent heat DSC – SOP Checking the DSC Firmware Version 3. From the DSC configuration window press the Firmware Version button. 4. The PyrisCfg window should indicate that the firmware version is version 6.4(or higher). WARNING : Do NOT press the update Flash EPROM button unless you are attempting to rewrite the system’s Firmware. Note : Older versions will report a phantom glass transition occurring at -127 o C during measurements (even when the sample pan is empty). This will cause a sudden discontinuity in specific heat measurements.

19. DSC outline – latent heat DSC – SOP Checking the Pyris Software Version 1. From the Windows desktop select : Start -> Programs -> Pyris software for windows -> Pyris Manager 2. An embedded Pyris manager toolbar will appear on the screen. Press the Pyris 1 DSC button.

20. DSC outline – latent heat DSC – SOP Checking the Pyris Software Version 3. From the Pyris Software main window toolbar, select : Help -> About… 4. The About Pyris window should indicate the software version as 3.72(or higher). Note : If the version is lower, you must update the software for proper operation

21. DSC outline – latent heat DSC – SOP DSC Gas Connections The DSC requires 3 types of gas connections for operation - Pressurizing gas - Shield gas - Purge gas All of the gases should pass through a drier system to remove moisture

22. DSC outline – latent heat DSC – SOP DSC Gas Connections - Pressurizing gas : The cryofill unit needs to be pressurized with Nitrogen gas. This will allow liquid Nitrogen to be introduced into the DSC coolant bath. Note : supplied gas pressure should be 40 psi

23. DSC – SOP DSC Gas Connections - Shield gas : The DSC deck needs to be supplied with Nitrogen gas which will act as a shield gas when the DSC slide cover is opened. GAS FLOW Note : There is a secondary pressure regulator between the drierite and the shield gas inlet (rear corner of DSC) which controls the amount of gas flow when the DSC slide cover is opened. Increase the flow If shield gas flow is inadequate to prevent external moisture and particles from entering the sample holders.

24. DSC outline – latent heat DSC – SOP DSC Gas Connections - Purge gas : The DSC chamber needs to be supplied with Helium or Nitrogen gas which will continuously purge the chamber of foreign substances. Note : supplied gas pressure should be 25 psi - Use Helium for scanning range of -170 ~ +250 o C - Use Nitrogen for scanning range above +250 o C - Use Ultra High Purity grade gases for purging

25. DSC outline – latent heat DSC – SOP Cryofill Liquid Nitrogen Filling Procedure In addition to the gases, the cryofill needs to be filled with LN 2 1. Open the Pyris software main window 2. Enable BOTH the shield gas and cover heater by pressing the buttons on the Pyris control panel. Depressed buttons are at the ON state. Note : make sure that the enable cryofill button is at an OFF state

26. DSC outline – latent heat DSC – SOP Cryofill Liquid Nitrogen Filling Procedure 3. Connect a liquid nitrogen supply tank to the cryofill inlet valve using a cryo line(hose) but do not open any valves yet. 4. Open the cryofill vent valve completely, but allow the valve to be loose and not completely be jammed in the counterclockwise direction. You may have difficulty closing the valve if you do so due to ice formation around the valve handle. 5. Open the cryofill inlet valve completely, but allow the valve to be loose as described previously.

27. DSC outline – latent heat DSC – SOP Cryofill Liquid Nitrogen Filling Procedure 6. Open the liquid nitrogen(LN2) tank supply valve slowly and observe the pressure buildup in the cryofill. Pressure around 9 psi is normal, but should not exceed 12 psi. If the pressure is too high, reduce the inflow of LN2 accordingly. You will observe nitrogen gas escaping through the cryofill vent during the filling procedure. When the cryofill is almost completely filled, LN2 will sporadically burst out from the vent. This is a good time to end the filling as described in the next step. Remember to protect yourself from LN2 freeze burns with cryo gloves and protective garments. 7. Close the valves in the following order : LN2 supply tank valve -> Cryofill inlet valve -> Cryofill vent valve 8. Loosen the cryo line(hose) to prevent pressure build-up inside the line(hose).

28. DSC outline – latent heat DSC – SOP DSC Liquid Nitrogen Filling & Equilibration The DSC needs to be filled with LN2 from the cryofill and stabilizeded prior to performing runs and measurements. 1. If you have not done so already, enable BOTH the shield gas and cover heater by pressing the buttons on the Pyris control panel. 2. Enable the cryofill button on the Pyris control panel. The ON state will indicate the cryofill button in a BLUE color. The cryofill will now start pumping LN2 into the DSC. It takes about 3 hours for the DSC to stabilize. Note : It is always a good idea to prevent the sample holder temperature from becoming subambient during the equilibration process. If the temperature is becoming too low, set the furnace to heat the sample holders manually by entering a number (25 o C is a good choice) in the input box of the control panel and then pressing the manual go to temp button.

29. DSC outline – latent heat DSC – SOP Preparing Samples You will need to insert your sample for analysis into the DSC using sample pans. A weight of ~10 mg is a good amount of sample for analysis. 1. Measure the sample pan lid (L). 2. Measure the sample pan (P). 3. Place the sample into the sample pan and measure the weight (P+S). Make sure the sample is distributed evenly along the bottom of the pan for best results. 4. Cover the sample pan with the lid and crimp, using the Perkin-Elmer crimp. Rotate the press handle until the press contacts the top of the lid. Rotate the press handle an additional 360 degrees to assure sealing without excessive stress and deformation to the sample pan. 5. Measure the total weight of the sample pan (L+P+S) 6. Determine the sample weight (S) by two methods : (P+S) – (P) and (L+P+S) – (L) – (P) 7. Make sure the above two values are within 0.1 mg. 8. Additionaly, prepare a reference sample pan with nothing inside it.

30. DSC outline – latent heat DSC – SOP Inserting Samples into the DSC 1. Open the DSC slide cover. The nitrogen shield gas should be flowing. 2. Remove the platinum sample holder covers using the DSC tweezer. 3. Insert the sample pan into the LEFT sample holder, and the reference (empty) pan into the RIGHT sample holder. Cover the holders with their platinum sample covers and close the DSC slide cover. Reference Pan goes here Sample Pan goes here

31. DSC outline – latent heat DSC – SOP Programming a DSC Run Program your run using the Method Editor 1. Select the Sample Info tab and enter basic info.

32. DSC outline – latent heat DSC – SOP Programming a DSC Run 2. Selecting the Initial State tab, enter the necessary information as shown. Initial temperature : temperature where your run will start. Usually 25 o C Y Initial : reference value of heat flow. Usually 0. Purge gas : Set as Helium @ 20 ml/min Equilibrate temperature : Usually 0.01 degC Equilibrate heat flow : Usually 0.01 mW Equilibrate wait time : Usually 15 minutes

33. DSC outline – latent heat DSC – SOP Programming a DSC Run 3. Selecting the Program tab, enter the steps your run will consist of. Your run will start with an initial isothermal condition. Decide how long it will be isothermal in minute durations.

34. DSC outline – latent heat DSC – SOP Programming a DSC Run 4. Press Add a step to include additional steps. Choose either Temperature Scan for changing the temperature or Isothermal for maintaining a temperature. If you choose a Temperature Scan step, input the target end temperature and the cooling/heating rate.

35. DSC outline – latent heat DSC – SOP Starting a DSC Run Use the Instrument Viewer window to monitor the DSC. The Y-axis displays the heat flow in milliWatts and the X-axis can display either the elapsed time or temperature. Wait for the temperature and heat flow to stabilize before initiating your run. It is a good idea to manually set the temperature to the initial temperature of your run, then wait for stabilization. Stabilization is normally assumed to have been achieved when heat flow oscillation is within 0.01 milliWatts. Start your run by pressing the DSC start button. The run will start soon.

37. END OF SLIDES

39. ADD: phase diagram (Glyc) PHASE DIAGRAMS Phase diagram (System Glycerol-H2O) Reference : Lane Ind Eng Chem 1925

40. ADD: phase diagram (ternary) TS Phase diagram (System NaCl-H2O-Glycerol) Reference : Shepard et al. Cryobiology 1976

41. ADD: phase diagram (ternary2) TS Phase diagram (Ternary system) Reference : Shepard et al. Cryobiology 1976

42. Prenuc vs non-prenuc – DSC+cryoscope CRYOMICROSCOPY Prenucleated vs. non-prenucleated freeze-thaw (1xPBS + 6M glycerol, DSC + cryomicroscopy) prenucleatednon-prenucleated