Tips for ITC Cell Cleaning

There are three factors that influence cleaning.

– Cleaning solution (whether it effectively removes dirt inside the cell)

– Frequency (how often the cleaning operation is repeated)

– Temperature (at what temperature to maintain the Thermostat after filling both cells with the cleaning solution)

The importance is considered to be in the order: cleaning solution > frequency > temperature. An overview is provided in the following sections.

Cleaning Solution

The basic principle of cleaning solutions is to dissolve dirt without dissolving the equipment.

The ITC cell uses Hastelloy C276, and in addition to this, plastic resins and stainless steel are used as supporting materials. When using a cleaning solution, always select one that does not corrode or dissolve these materials. For solutions other than the recommended 14% DECON90 (or 20% Contrad70), some level of the above risks are involved.

For typical sample measurements involving proteins or nucleic acids, most dirt can be handled with the recommended 14% DECON90 solution. Additionally, in most cases, a new device or a device regularly maintained can be handled with 14% DECON90.

However, depending on the degree and type of dirt, cleaning with DECON90 may not efficiently remove it. For example, customers have recently been using highly hydrophobic low-molecular compounds as samples. In cases where dirt on the cell becomes an issue, cleaning with a 50% DMSO solution has been found to be effective. Choose cleaning solutions such as those listed below according to your objectives.

– 14% (v/v) DECON90 aqueous solution

– 1-5N NaOH aqueous solution

– 50% (v/v) DMSO aqueous solution

– Diluted household dish detergent

– 2-fold diluted pipe cleaner (Johnson & Johnson Pipe Unish)

– 1% (w/v) citric acid aqueous solution

– Detergents with proteolytic enzymes

Frequency

Even with the same cleaning solution and soaking conditions, repeated cleaning may be necessary for complete removal. Especially when dirt has accumulated, a single cleaning may be insufficient. Measure the cleaning effect using water-water titration, and repeat the process several times with an effective cleaning solution until the baseline and titration thermogram are restored to a good condition.

Temperature

Just like with household detergents, heating the cleaning solution during cleaning enhances its effectiveness against stubborn dirt. Under high-temperature conditions, there is a risk of corrosion or other damage to the cells, so always check the chemical resistance of the cell unit to the chemicals used, based on documentation, etc. Information on the materials (metals, resins) that make up the cell unit is provided in the appendix of the user manual (English) that comes with the equipment. If unsure, please inquire.

The following are specific cleaning methods.

Note Before Operation

– The cleaning solutions may be highly alkaline or hazardous. Always wear appropriate protective gear during operation.

– Extended soaking carries the risk of forgetting to rinse, leading to solution condensation, precipitation, and dirt solidification.

In the worst-case scenario, there is a risk of irreversible damage to the cell. Please carry out operations at your own responsibility.

– Depending on the state of the dirt, perform solution exchange for a maximum of about an hour. Absolutely avoid overnight soaking.

Items to Use

– Cleaning solution

– Gas-tight syringe

– Cleaning Device (for VP-ITC customers. A cleaning tool that allows solution delivery into the sample cell using the ThermoVac attached to the equipment)

– Cosmetic syringes (cosmetic subdivision syringes available at 100-yen shops. The tip is flatly cut, and 20 mL ones are easy to use.)

About Cleaning Solutions and Conditions

– 14% DECON90 aqueous solution (effective for proteins and other dirt, recommended cleaning solution for regular maintenance. Maximum soaking temperature 60°C, do not exceed 1 hour)

– 5N-NaOH aqueous solution (effective for proteins, peptides, nucleic acids, etc. Maximum soaking temperature 60°C, do not exceed 1 hour)

– 50% DMSO aqueous solution (effective when hydrophobic low-molecular compounds are used. Maximum soaking temperature 50°C, do not exceed 30 minutes)

Other documented cleaning solutions:

– Diluted household dish detergent (diluted about 2 to 5 times. Effective for lipids and other oils. Look for words that claim to remove oil stains, maximum soaking temperature 50°C, do not exceed 30 minutes)

– 2-fold diluted pipe cleaner (for colored substances, dirt, proteins, etc., use Pipe Unish Pro, maximum soaking temperature 50°C, do not exceed 30 minutes)

– 1% citric acid (dirt dissolved in acid, for samples containing Ca, Mg, etc., in the buffer, maximum soaking temperature 50°C, do not exceed 30 minutes)

– 1% Zymit (cleaning solution with proteolytic enzymes. Purchase the concentrated solution and dilute to the concentration specified by the manufacturer. Maximum soaking temperature 25°C, do not exceed 12 hours)

Cleaning Operations

1) Remove ultrapure water from both the Sample/Reference cells. Remove the Reference cell plug.

2) Use a gas-tight syringe to fill both cells with cleaning solution (in the same manner as filling the cell with a sample).

3) Slowly perform a pumping operation to allow the cleaning solution to penetrate the entire cell.

4) Discard all the cleaning solution from both cells once.

5) Refill both cells with cleaning solution (remove as much excess cleaning solution as possible. This is similar to when filling with a sample).

6) Set the cell temperature to the target temperature.

7) Cover the cell ports with a paper towel, etc., to prevent dirt from entering the cells.

8) Depending on the state of the dirt, soak for 10 minutes to a maximum of 1 hour.

9) Once the soaking is complete, return the cell temperature to 25°C (Note: if you insert a gas-tight syringe while the cleaning solution is warm, the syringe may be damaged, or contents may splash out from the cell).

10) Carefully extract the cleaning solution that has returned to room temperature using a gas-tight syringe, then immediately rinse the gas-tight syringe thoroughly with ultrapure water.

11) Use a gas-tight syringe to fill both cells with ultrapure water and slowly perform a pumping action. Move it up and down several times to rinse the entire cell.

12) Rinse each of the Sample/Reference cells with approximately 50-100 mL of ultrapure water in total. For the Sample cell, cleaning with a Cleaning Device is convenient.

13) Cleaning solution may adhere to the cup part of the Sample cell and the part where the Reference plug is inserted. Please rinse as appropriate.

14) When inserting the needle part of the gas-tight syringe into the cell, do so slowly. If the metal tip hits the bottom of the cell with force, it may scratch the cell surface, causing noise and, in the worst-case, malfunction. When rinsing, inject only enough to avoid overflowing from the cell port.

15) Once rinsing is complete, fill both cells with ultrapure water.

Precautions During Cleaning Operations

– Please ensure both cells are rinsed thoroughly with ultrapure water. For the Sample cell only, using the ThermoVac/Cleaning Device is also effective.

However, as the Reference cell cannot be rinsed at all, please be sure to rinse the Reference cell in the same way.