1234 Hach Hall
515-294-5805
[Link]
Microcal VP-ITC Operating Instructions
10/18/2012 S.V. draft
Location: 1238 Hach Hall
Contact: Steve Veysey, 1234 Hach Hall
SAFETY
All researchers working in 1238 Hach Hall must complete the EH&S course: “Fire Safety and Extinguisher
Training”. When preparing samples in this room, please wear all appropriate personal protective equipment.
Aprons, safety glasses, and rubber gloves are available in 1238A Hach Hall. All use of solvents should occur in
1238A Hach Hall.
Properly dispose of glass pipettes and plastic pipette tips in the containers provided. Waste solvents can be
disposed of in the waste containers provided in 1238A. All of the computers in this lab have direct links from
the desktop to MSDS sheets, the EH&S Laboratory Safety Manual and to the CIF Safety Manual.
INTRODUCTION
The MicroCal VP-ITC (Isothermal Titration Calorimeter) unit directly measures heat evolved or absorbed in
liquid samples as a result of mixing precise amounts of reactants. A spinning syringe is utilized for injecting and
mixing of reactants. The user inputs the experimental parameters (temperature, number of injections,
injection volumes) and the computer carries out the experiment. Origin® software is then used to analyze the
ITC data using fitting models to calculate reaction stoichiometry (n), binding constant (KD), enthalpy (ΔH) and
entropy (ΔS).
This document presents a training guide prepared in 2006
by former ISU student Dr. Gulden Camci-Unal. Minor
procedural changes are annotated and summarized at the
end.
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Note: Pick YOUR
username from the ITC
users list
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Reminders:
• Report all problems with the instrument to Steve. Make an entry in the logbook, but follow up with an
e-mail to sveysey@[Link].
• Handle syringes carefully; be careful not to lose the small stir bars.
• Updated versions of manuals, technical notes, et cetera can be found at our website
[Link].
• You must properly complete an entry in the logbook each time you use the instrument.
• You may reserve up to 12 usage blocks per month without approval. Additional blocks must be
approved by Steve.
• Scheduling mistakes should be neatly crossed out with one line so that the original name is still legible.
Send an email to Steve explaining why you will not be using the time you have scheduled. You may be
charged for scheduled but unused blocks.
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APPENDIX A – VP-ITC TRAINING TUTORIALS
After reviewing Guldens training guide, you must successfully complete the three practical measurements described
below. NOTE: You must also complete the Origin Data Processing – Lesson 1 available at our website. This will ensure
that you have developed sufficient expertise to use the ITC properly without causing problems for other users.
Tutorial 1. Water to Water Baseline
1. Log on to the VPC-ITC control computer.
2. Check the logbook to see when the water in the reference cell was last replaced. If it has not been replaced
within the past month, contact Steve and arrange to have the reference cell solution refreshed.
3. Degas approximately 20 mls of distilled water for at least five minutes. This degassed water will be the solution
that will go into the sample cell and the syringe.
4. Load your 2.5 ml glass-filling syringe with water. Remove air bubbles. Insert the long needle into the sample cell
entry tube (center tube) and carefully slide it down until it touches the bottom. Lift it up slightly and slowly
depress the plunger to so that the cell fills from the bottom up. When you see the water level reach the top of
trhe narrow access tube, depress the syringe plunger quickly 1-3 times to deliver abrupt bursts of about 0.25 ml.
This will dislodge small bubbles that may have attached near the bottom of the cell.
5. Slowly withdraw the syringe until it is resting on the lip at the top of the sample cell entry tube. Carefully suck up
any water that is in the funnel area just above the entry tube.
6. Launch the VPViewer application. <logon as your account>
7. Call up the water-water parameter set <Load Run File; select [Link]; Open>
8. Enter a filename for the water-water data you are about to acquire. No spaces, periods or hyphens; maximum of
16 characters.
9. Verify that the VP-ITC thermostat temperature is set to the desired run temperature of 25 degrees.
10. Load the ITC pipette. Place the water titrant into the pipette filling tube, then place the filling tube into the
bottom of the pipette stand. Carefully insert the auto-pipette into the pipette stand. Be very careful not to hit
the long needle of the injection syringe against any object. The needle must not be bent.
11. In the software, select the ITC controls window. Click on the Open Fill Port button. The fine-thread screw
controlling the syringe plunger should be observed to turn, and the plunger should move upwards until the
white Teflon plunger tip is positioned just above the fill port.
12. Attach the tube of the plastic filling syringe to the filling port of the injection syringe.
13. Slowly withdraw the plunger of the plastic filling syringe to draw up the titrant solution until the solution begins
to enter the filling syringe. Clicl on the Close Fill Port button. The injection syringe plunger tip should move to
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just below the filling port orifice. Remove the hose of the plastic filling syringe from the filling port of the
injection syringe.
14. Click on the Purge->Refill button. The auto pipette screw will depress the plunger filling syringe, then draw it
back up again. This will help dislodge air bubbles that may be in the syringe. Repeat the Purge->Refill process
two more times.
15. Carefully remove the pipette from its stand by picking it straight up until the glass barrel of the injection syringe
is above the top part of the pipette stand. Carefully move the pipette so that it is directly above the center-
positioned sample cell access tube. Insert the pipette into the sample cell access tube by holding the pipette
vertically and slowly lowering the pipette. When the pipette is almost completely inserted you may have to
push down slightly to compensate for the resistance of the rubber o-ring so that the pipette is properly seated.
16. Select the START button to begin the experiment. You may receive a query about “Invalid Cell or Syringe
Concentration”. This is irrelevant for the water-water titration. Click Yes to continue.
The entire run will take approximately 30 minutes. The results should be similar to the graph shown below.
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Tutorial 2. Methanol to Water Titration
A methanol injection run requires a higher level of precise preparation and operation of the VP-ITC instrument than the
water-to-water- run.
Follow the procedures recommended in the water-to-water tutorial for preparation of solutions, degassing, filling the
cell and injection syringe, et cetera.
Sample Preparation
o Fill the sample cell with degassed water.
o Prepare 20 ml of degassed water in a glass vial and add 0.5 ml of methanol to make a 2.5% methanol
solution.
o Degas the methanol solution (with stirring) for 5 minutes.
o Load the methanol solution into the injection syringe of he pipette.
Conducting the Experiment
o Load the parameters from the [Link] file. <Load Run File; select [Link]; Open>
o Enter a filename for the methanol-water titration data.
o Execute the run and allow the system to complete the experiment.
Analyzing the Data
o After the run is complete, minimize VPViewer.
o Open the separate Origin 70 program for data analysis. Read in the data you have just acquired. The raw
ITC data should look like the results in the figure below.
o View the data in the DeltaH window. Generate a linear regression fit; the first data point should be
excluded. The DeltaH data should look like the results in the figure below.
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Tutorial 3. EDTA – CaCl2 Titration
The final tutorial will require that you are familiar with the more advanced data processing techniques available in the
Origin 7 software. Nine Origin Data Processing lessons are available at our website. You must complete Lesson 1
“Routine ITC Data Analysis and Fitting” before you will be able to complete this tutorial.
Sample Preparation. Obtain a test kit labeled “EDTA – CaCl2 Titration” from the refrigerator. Each kit will contain
o A tube of EDTA solution labeled with concentration and buffer conditions
o A tube of CaCl2 solution labeled with concentration and buffer conditions
o A vial of 10 mM MES buffer solution for rinsing the cell prior to sample loading
IMPORTANT NOTE: The control titrations for this experiment will not be performed. The system will be going to
saturation and the small, repeatable peaks at the end of a well designed experiment are a very good representation of
control heats.
Setting Up the Experiment. Load the parameters from the [Link] file. <Load Run File; select [Link]; Open>
Note: The instrument should be thermostatted at 25oC to facilitate a faster start.
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Total # Injections 29 Volume 1 Injection 2
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Cell Temperature 25 Duration 1 Injection 4
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Reference Power 10 Volume after 1 Injection 10
st
Initial Delay 60 Duration after 1 Injection 20
Syringe Concentration 1 Injection Spacing 210
Cell Concentration 0.1 Filter Period 2
Stir Speed 307 Feedback Mode/Gain High
ITC Equilibration Options Fast Equil; Auto
NOTE: Cell and syringe solution concentrations need to be entered manually in all cases.
Loading Samples
1. Rinse the sample cell with 10mM MES buffer two times
2. De-gas both the EDTA as well as the CaCl 2 samples for 5 minutes only
3. Load 2ml of 0.1mM EDTA into sample cell; pipette the liquid up and down for several times and soak for at least
2 minutes
4. Empty the sample cell, and reload fresh EDTA for the experiment
5. Clean the injection syringe with methanol and dry thoroughly before loading 1.0mM CaCl 2
Analyze the Data
Analyze the data in Origin and compare the results to those on the graph below. If the results are within the
specifications stated below, the instrument (and the operator) is performing correctly and you are ready to run
experimental samples.
The results which you obtain should be very close (i.e. N value +/- 5%; K value +/- 20 %; and Delta H value +/- 10%) to the
values shown in the results at the end of this tutorial
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Expected Experimental Results
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It should be noted that no control heats were subtracted during the analysis of the attached results and that the same
procedure should be applied to your results. It should also be noted that the experimental energies for the peaks were
obtained using the automated integrations within Origin, but that in some cases those automated integrations might
need to be manually adjusted to get agreement of the results.
Common Problems – What to Do?
If your results do not agree with the expected results then you might want to repeat the experiment to see if your
results are reproducible, or not. In repeating the experiment, consider the following possible causes of bad results:
1.) Sample dilution will affect your results and specifically will change the resulting ‘N’ value obtained from the fit of
your binding isotherm. Dilution of the cell solution (EDTA) will lower the ‘N’ value and dilution of the titrant
solution (CaCl 2 ) will increase the ‘N’ value. The net change in ‘N’ will be the difference in dilutions between the
cell and titrant solutions. Avoid dilution factor by using completely clean and dry transfer syringes, and also by
pre-soaking the cell with EDTA.
2.) Sample contamination from the MeOH can cause for bad experimental results. Be sure to thoroughly dry the
MeOH from the injection syringe prior to loading it with CaCl 2 .
3.) A dirty sample cell or a dirty injection syringe can affect your results and also the quality of the data. Be sure to
thoroughly clean the sample cell and injection syringe prior to carrying out the experiment with the standard
sample kit.
4.) If problems persist then e-mail labcrew-us@[Link] and specify your instrument model and be sure to attach
the raw data files that you generated (*.itc files). We will review your results and provide you with an
assessment as well as any pertinent suggestions for improvement.
APPENDIX B ORIGIN 7 LESSONS
You must complete Lesson 1 before attempting to use the VP-ITC. THe raw data files associated with the lessons can be
found at:
All of the lessons are posted as PDF files at our website, [Link].
Lesson 1 Routine ITC Data Analysis and Fitting
Lesson 2 Setting Baseline and Integration Range
Lesson 3 Deleting bad data
Lesson 4 Analyzing Multiple Runs and Subtracting Reference
Lesson 5 ITC Data Handling
Lesson 6 Modifying Templates
Lesson 7 Advanced curve Fitting
Lesson 8 Autosampler Data (optional accessory – we do not have it)
Lesson 9 Other Useful Details
Lesson 10 Equations Used for Fitting ITC Data
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