If you have problems, find a Microanalysis staff member. If no one is around or it is after hours call Danielle Gray 630-865-5844. 

All users must be trained to run experiments.

Before you can be trained:

• Obtain a set of blunt tip needles and syringe loading adapter for your lab.
• Optional, obtain your own glass loading syringe.
• Watch YouTube video on how to manually load titrant syringe.
• Watch Webinar on Introduction to Characterizing Binding via Isothermal Titration Colorimetry (ITC)
• Download NanoAnalyze for yourself from TA Instruments.
• Fill out and have your PI sign the training permission form. 
• Schedule training with a Microanalysis Technician. 

Training will consist of at least 3 sessions:

1. You must successfully perform a water to water titration to demonstrate good technique. 
2. After a successful water to water titration, you will do CaCl₂ into EDTA titration to see a binding and data analysis.
3. You will perform your first experiment with a Microanalysis Technician. We will discuss your solutions and experiment and make sure that the default cleaning parameters are acceptable or if we need to develop a new protocol for your experiments. 
4. Any time your experiments are deviating from what has been an approved use of the ITC, you must discuss the experiment with the technicians. 

Starting an ITC experiment (AQUEOUS MODE SOP)

1. Look at similar systems and decide a starting concentration range for your sample and titrant. 
   1. All materials MUST be in solution. 
   2. All chemicals must be approved for use in the instrument by a Microanalysis technician. 
   3. Prepare any additional cleaning solutions outside of the 3 provided
      1. Di-Water
      2. 1% Contrad Soap solution
      3. Methanol
   4. Prepare extra titrant and solution buffers for conditioning of the instrument.
2. A clean workspace is a happy workspace so replace the large kimwipe for a new clean work surface. 
3. Make sure:
   1. There is enough Di-Water, soap solution, methanol, or other syringe washing solutions for your planned experiment.
   2. There is enough solution in the cleaning station as well.
   3. The Waste flask for the cleaning station is empty. 
   4. The Waste container for the syringe wash is not full.
   5. When possible, degas sample solutions prior to placing them in the Affinity ITC in order to minimize the possibility of gas bubble formation during the run. 
4. Rinse all the needles, adapters, and syringes with di-water prior to use.
5. Log into ChemFOM. 
6. Open ITC. Run the software that we run experiments in. 
7. Create your experiment in the software in the Experimental Method Tab. 
   1. RED BOX
      1. it can be adjusted 75-150 RPM but discuss with a technician prior to changing
      2. The temperature is 25°C
         1. This can be changed but discuss with a technician prior to changing for training. 
         2. 2-80°C effective temperature range, but additional steps are needed to protect cell if temperature is changed from 25°C. 
   2. GREEN BOX
      1. Type in your syringe titrant concentration in mM
      2. Type in your Cell Concentration in mM. If none for blank, type 0.001
   3. BLUE BOX
      1. Decide what type of experiment you want to run.
         1. Incremental Titration (described in the rest of this SOP)
         2. Continuous Titration (see Continuous Titration SOP)
      2. Setup
         1. Pick injection intervals
         2. How many injections
         3. Quantity of solution injected (remember this is a low volume ITC 1-3uL/injection is normal for about 50 μL total)
      3. Make sure you choose the auto equilibrate based on the expected heat of your reaction. If you do no know, choose medium heat.
      4. Choose an initial and final baseline that should match the time of your intervals.
   4. ORANGE BOX
      1. Select a syringe cleaning method if you are going to load enough titrant for one experiment. If you are going to overload the titrant syringe with titrant for more than one reaction, do not select a cleaning method until your last experiment.
         1. Default cleaning method is 2 mL soap, 10 mL di-water, 10 mL methanol.
         2. If you think your materials need a different cleaning method, verify cleaning method with staff and then create your own based on the chemicals you are using. Once verified, you can load your saved cleaning method. 
8. The instrument has a Sample Cell and Reference Cell. The reference cell is on the right and is filled with a volume of high purity di-water. This is done by a Microanalysis  Technician and is refilled every 2-3 weeks. See instrument book for exact quantity loaded so you can load the same volume into the sample cell. 
   1. Use a long blunt tipped needle and remove the reaction cell solution from the instrument. When inserting the long needle into the cell, be careful not to scratch the bottom of the cell. The needle should be inserted slowly, gently, and straight into the instrument. When you feel the needle touch the bottom of the cell, lift up a few mm and begin filling or sucking out the solution.
      1. If the mixing arm is in the reaction cell, press the Park Arm button so it returns to the filling and cleaning position. 
      2. Press the flashlight button to light up the cells so you can see.
      3. There should be 300μL of DI-water in the reaction cell or your previous reaction material.
   2. Wash the cell 3 times with sample to condition the cell and remove any trace of previous sample. Suck up an re-inject the solution 2 or 3 times before you discard and aliquot in the waste beaker. 
   3. Finally, on the 4th time, fill the cell with the same amount of solution as the reference cell (again, see the instrument book for exact quantity). Inject evenly and swish tip of needle as you remove it to make sure there are no bubbles in the reaction cell. 
9. Rinse the syringe with DI-water.
10. Now you are done with the experimental setup in the software and are now going to use the WIZARD to load your titrant. Press play and a wizard will open. 
    1. Follow the directions in the WIZARD. DO NOT SKIP AHEAD.
    2. If you need to load titrant into the syringe, click Refill Syringe and then click Next.
       1. Note: if this is a continuation of a previous experiment or you are using the remaining titrant from a previous loading, select "Use Remaining Titrant" and follow the rest of the instructions for the wizard. You can load up to 250μL of titrant, but you only use ~50μL per experiment so you can use the loaded titrant for more than one experiment. 
    3. The instrument will move the syringe and arm if necessary. Wait and be patient. Do not skip ahead. Obey all WIZARD prompts. The next screen will be: 
    4. Follow the directions. (Note: The loading syringe is stored in the Styrofoam box; the injection/titrant syringe is attached to the instrument and is not to be removed.)
       1. Load 300μL of blank buffer that matches the titrant buffer. (Note: make sure to rinse syringe with buffer before the final load.)
       2. Attach the adapter to loading syringe. 
       3. Unscrew canula line at top and screw in the adapter/loading syringe assembly. (See red circle in picture.)
       4. Inject the buffer.
       5. Remove the loading syringe and fill with air.
       6. Re-attach to adapter and push the air through.
       7. Then remove the loading syringe and press Next.
       8. Rinse the loading syringe with your titrant. 
    5. The next screen will be:  
       1. Fill syringe with at LEAST 100 μL of titrant (this also depends on your experiment, you may need more). Make sure that you have loaded enough titrant for your experiment. 2.5 μL x 10 titrations = 25 μL plus the volume needed to fill the cannula (~50 μL) and err on the side of excess. At MOST 250μL of titrant can be loaded.
    6. The next screen will be: 
       1. Reattach the loading syringe to the adapter. Check that the adapter is securely screwed in to avoid leakage. 
       2. Fill the port with aliquot of titrant. 
       3. Make sure there are no bubbles or breaks in the titrant (injection) syringe barrel. 
       4. Leave syringe and adapter attached until you are instructed to remove the assembly. 
    7. The next screen will be:  
       1. Type the total amount you loaded into the titrant syringe. 
       2. Leave assembly attached until you are instructed to remove.
       3. Next and it will move the plunger.
    8. The next screen will be: 
       1. Remove the plunger
       2. Reconnect the cannula fitting to the port. Finger tight only.
       3. Click finish and name your experimental file.
          1. Save the file in your folder that is within your PI folder.
          2. Date, notebook code, and other necessary identifier tags. Example: 06032020_water_into_water.
    9. The titration arm will move from the loading station into the titration cell and the experiment will begin. It will first need to equilibrate and once it equilibrates, it will tell you your experimental time in the top bar of the ITC run window. You can watch the data collect in the Data tab. 
    10. After starting the experiment: Now clean up time. 
        1. Rinse all your needles, syringe, and adapter.
           1. Use 4-10 aliquots of DI-water and methanol.
    11. In the logbook, identify the date, your name, and the chemicals you used in the instrument. This will help keep track in case there are issues and questions about cleaning.
        1. Unsure of what can and can't be used in the instrument? ASK a staff member. 
    12. Stay and watch your experiment or come back at least 5 minutes before your experiment should be done. 
    13. If you ran a syringe cleaning cycle after your experiment (7.d), the titration syringe arm will be back in the standby location. If you were going to use the same titrant immediately for another experiment, then the titrant arm will be in the experimental configuration. To move the arm back to the standby position, press the park arm button. 
    14. Your experiment is over. Now clean the sample cell.  
        1. Manual clean (Note: do not do this unless it is cleared with a technician and you have been shown how.)
        2. Cleaning Station (Preferred) See Cleaning Station SOP. 

CLEANING STATION SOP

1. Make sure the water (full 2L), soap (200mL or more) and methanol bottles (200mL or more) are full enough. 
2. Remove the old sample with long needle. If you cannot see, press flashlight button. When inserting the long needle into the cell, be careful not to scratch the bottom of the cell. The needle should be inserted slowly, gently, and straight into the instrument. When you feel the needle touch the bottom of the cell, lift up a few mm and begin sucking out the solution. 
3. Close ITCRun because you are going to be putting large amounts of solution through the instrument and do not need constant monitoring. 
4. Inspect the boot of the cleaning tool for tears or rips that would prevent it from creating a vacuum. 
5. Place the cleaning station tool into the instrument slowly and gently. The 4 notches should match up. The height is already adjusted so as not to scratch the cell. Do not adjust the height of the tool. 
6. Make sure the waste flask is empty and the hose end is pointing away from the vacuum arm. 
7. Touch the cleaning station touch pad. DO NOT wear gloves when touching the cleaning station control panel. 
8. Use the saved default cleaning method unless you have permission from the staff to use a different reaction cell cleaning method based on your reaction chemicals. 
   1. Default_Micro_Clean method
      1. 10mL 1% Contrad detergent solution (solvent 2 in station) 
      2. 1L Deionized water (solvent 1 in the station)
      3. 10 mL Methanol (solvent 3 in the station)
      4. Air for 5 minutes
9. Select the cleaning method and hit start then hit play. Watch the cleaning cycle and make sure the liquid level in the flask does not reach above the blue line (arm). If it gets too close, then pause the cleaning cycle and dump waste into marked containers. Re-stopper and resume with the play button. This should take about 15-20 minutes. 
   1. If you had to pause notify a Staff member so they can fix the flow rate calibration. 
10. Remove the cleaning plunger.
11. Using a clean, long needle place ~300μL of deionized water into the reaction cell so the instrument is in standby for the next user or use refill process for reaction cell for your next reaction [8).a-c of Starting an ITC experiment (AQUEOUS MODE SOP)]. When inserting the long needle into the cell, be careful not to scratch the bottom of the cell. The needle should be inserted slowly, gently, and straight into the instrument. When you feel the needle touch the bottom of the cell, lift up a few mm and fill the cell with solution. 
12. NEVER LEAVE THE REACTION CELL DRY.