MicroCal PEAQ-ITC with organic solvents: Tips and cautions

Summary

There is an increasing interest in Isothermal Titration Calorimetry (ITC) applications that require dissolving the binding partners in an organic solvent. These include guest: host chemical reactions, nanoparticles, and insoluble materials. For successful results, the experimental protocol with organic solvents requires minor modification of the normal methods with aqueous buffers. This blog includes tips on performing successful experiments with organic solvents on a MicroCal PEAQ-ITC system.

If you are using another MicroCal ITC, or have more questions, contact your local Malvern Panalytical office or the help desk.

Before you use organic solvents with a MicroCal PEAQ-ITC or another MicroCal ITC system, please review these cautions prior to any experiments:

• Malvern Panalytical has not tested all instrument components with all organic solvents.  You can get a complete list of wetted surface components from the MicroCal PEAQ-ITC users guide, or your regional Malvern Panalytical office (a partial list is below). MicroCal VP-ITC and iTC200 use some different materials.
• Check chemical resistance guide(s) for more information on specific instrument components and solvent(s) of interest. There are several guides available online.
• Limit the time the PEAQ-ITC is exposed to organic solvents,  and remove organic solvents completely after use. Do not keep organic solvents in ITC cells or syringes. Clean any spills immediately.
• It is possible for volatile gases from solvents to seep into instrument electronics, and damage the cell core. This is an expensive repair.
• Do not use organic solvents with PEAQ-ITC wash module, or the autosampler for the Automated PEAQ-ITC, unless the solvent is known to be compatible with ALL instrument components.
• Know the human and environmental risks of the organic solvents, and preventive measures. Use organic solvents in a well-ventilated laboratory, and appropriate personal protective equipment.  Discard organic solvent waste by appropriate methods.

Here is a list of solvents known to be compatible with MicroCal PEAQ-ITC:

• Aqueous buffers between pH2-pH12
• 1 M NaCl
• 50% DMSO
• 50% acetonitrile
• 100% Methanol
• 50% Ethylene glycol
• 20% Contrad 70
• 14% Decon 90

Partial list of wetted surface materials on MicroCal PEAQ-ITC – Check chemical resistance guides for specific solvents. This list does not include all of the wetted components of the washing module,  and all of the materials used in the PEAQ-ITC autosampler.

• Hastelloy alloy C276: ITC cells
• Acetal (also called Delrin): ITC cell port overflow cups
• Stainless Steel 316: Injection syringe, manual cell loading syringe, ITC cell ports
• Stainless Steel 303: Injection syringe nut
• PEEK: Fill port adaptor (FPA), washing module
• Kalrez: Fill port adaptor (FPA)
• PTFE: Injection pipette, injection syringe bearing sleeve, manual cell loading syringe
• Borosilicate glass: Injection syringe, manual cell loading syringe
• Tygon 2375: Tubing for washing module and FPA assembly
• Polypropylene: Quick connect fittings for washing module and Fill Port Adaptor (FPA) assembly
• FEP (fluorinated ethylene propylene): sheath for manual cell loading syringe
• NOTE: some organic solvents can soften the 0.2 mL tubes used to fill the injection syringe, and degrade the adhesive used to assemble the manual cell loading syringe.

How to perform experiments with organic solvents

Sample preparation with organic solvents:

• Each binding partner for the ITC titration needs to be in identical solvent. Any mismatch due to another solvent or additive will result in large background heat.
• Use dry solvents, supplied in sealed ampoules, or dried with a molecular sieve. This will reduce water contamination during the ITC experiment.
• Sample should be pure, with no extra materials, additives, solvents, etc.
• Carefully weigh out samples and mix with solvent. Use immediately to prevent concentration changes, contamination by moisture, etc.
• If you need to replace one solvent with another prior to ITC, be sure all the initial solvent is removed from sample via appropriate method (i.e. lyophilization).
• If you are using a mixed solvent (i.e.  50% acetonitrile/50% water): these are very difficult to exactly match. Be sure you perform appropriate control titrations.
• Degassing samples prior to ITC may not be effective for organic solvents. Be careful about potential outgassing when filling the ITC cell and syringe
• If you are using a volatile solvent, note that the sample concentrations in the ITC cell and syringe will likely change during storage, and during the ITC experiment, due to solvent evaporation

Preparation of MicroCal PEAQ-ITC cells and injection syringe for experiments with organic solvents

Before 1^st  experiment:

• Clean the sample and reference ITC cells and injection syringe with 20% Contrad 70 (or 14% Decon 90), followed by several water rinses to remove proteins and other contaminants. Repeat cleaning if needed. Remove water.
• Rinse the ITC injections syringe with methanol, then dry with PEAQ-ITC pump to remove methanol.
• Rinse sample and reference  ITC cells with methanol, using manual cell filling syringe.  Remove methanol.
• With the ITC cells empty, thermostat to 60°C, for 30-60 minutes to evaporate methanol. This will dry the ITC cells.
• Thermostat ITC cells to experimental temperature.
• Fill reference cell with experimental solvent (do not use water)
• Fill  sample cell with sample in solvent.
• Fill injection syringe with sample in solvent

Between experiments with the same solvent:

• Remove sample for sample cell with manual cell loading syringe. Rinse cell with solvent several times, using manual cell loading syringe. Fill sample cell with new sample + solvent.
• Rinse the ITC syringe several times with solvent. Use Fill Port Adaptor connected to syringe, bypassing  PEAQ-ITC washing module. Fill syringe with sample + solvent. 
• Replace solvent in reference cell if evaporation has occurred.

After final experiment in organic solvent:

• Remove solvent solution from sample and reference cells with manual cell loading syringe. If solvent is not miscible with water, rinse ITC cells several times with methanol with manual cell loading syringe. Follow with water and detergent solution wash.  Rinse with water and leave fresh water in sample and reference cells.
• If solvent is not miscible with water, rinse the ITC syringe several times with methanol, then water and detergent solution. Rinse with water and methanol, store syringe dry
• If solvent is not miscible with water, rinse the manual cell filling syringe several times with methanol, then water solution. Store syringe dry
• Make sure any organic solvent spills on the instrument are cleaned up

Suggested experimental design and control experiments

• For MicroCal PEAQ-ITC: 25 °C, 19 X 2 µL injections, 150-sec interval, 500-750 rpm stirring (suggest stirring speed below 1000 rpm for organic solvents).
• May need slower stirring speed to reduce noisy baseline between injections, and/or different temperature
• If baseline shifts, this may be due to bubbles in ITC cell and/or syringe
• It is important to perform several control ITC titrations,  to ensure that the observed heat changes are due to binding, not another process, or from solvent mismatch
  • Water titrated into water: this is the recommended test for overall ITC function
  • Solvent titrated into solvent
  • “Ligand/guest” + solvent in ITC syringe, titrated into solvent only in ITC cell
  • Solvent only in ITC syringe, titrated into “macromolecule/host” + solvent in ITC cell
  • All of these control experiments should give small, reproducible changes per injection, with low noise
• If heat changes per injection are larger than expected for controls and/or experiments, look for sources of moisture contamination and solvent mismatches, and make sure the ITC cells and syringe were cleaned.
• If there is no evidence of binding and/or binding saturation, also check concentrations.

Example: Host – guest interaction in Acetonitrile

• Raw ITC data (top) and data fit to one set of sites model (bottom) for host-guest recognition reaction in acetonitrile
• “Guest” in the syringe at 17.5 mM and “host” in the cell at 0.7 mM.
• Experiments performed with MicroCal PEAQ-ITC, analyzed with PEAQ-ITC software.

Further reading:

• Isothermal Titration Calorimetry: Theory and Practice
• Top Ten Factors to consider when buying an Isothermal Titration Calorimeter
• MicroCal PEAQ-ITC user manual
• MicroCal PEAQ-ITC operating instructions