pH Measurement How to Measure pH in Small Samples Accurately?

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Mettler-Toledo GmbH

KNF Neuberger GmbH

Small sample volumes — a common situation in laboratories. This can cause problems when measuring pH. Various problems can be solved by using specific sensors.

In life sciences, many applications necessitate the analysis of small samples, often requiring multiple testing protocols. These limited-volume samples, representing valuable experiments, are susceptible to errors introduced during sample manipulation. However, an appropriate pH sensor and the right measurement practices, can significantly reduce the required sample size and the extent of manipulation needed, thereby enhancing the accuracy of the results.

Some of the common small-volume applications include:

• RNA sample analysis through microarrays,

• DNA sample analysis via PCR,

• Protein sample analysis using immunoprecipitation.

So, why is it challenging to measure pH in small samples? It can be tricky because of three main factors: the sensing glass, the reference junction, and the temperature. The sensing glass is the part of the pH sensor that interacts with the solution. It’s designed to generate a voltage when it comes into contact with hydronium ions. For accurate readings, the sensing glass needs to be fully immersed in the sample. That’s why regular-sized sensors don’t work well for tiny samples — they often leave the sensing glass partly exposed, which leads to errors.

The reference junction, located just above the sensing glass, is essential for allowing the reference electrolyte to flow into the sample. If small samples don't reach this junction, the electrolyte flow stops, and the readings can be inaccurate.

Also, pH results are only correct if the sample temperature is taken into account during the measurement. When working with very small samples, their mass is tiny compared to the sensor. This means the sensor might take a long time to reach the right temperature. If the sensor’s temperature isn’t accurately matched to the sample’s temperature, the pH reading can be off. To get reliable results with small samples, it's important to use the right sensor with an internal sensor temperature probe. Fortunately, there are sensors designed to measure pH in small samples directly without diluting them.

Micro sensors

Micro sensors include the same components as a standard pH sensor, configured to optimize measurements in small containers. Whereas a standard sized sensor requires a few milliliters of sample to measure accurately, a micro sensor has miniaturized components and can be successfully used in much smaller samples.

Mettler Toledo has multiple sensors that have a micro membrane that is hemispherical — InLab UltraMicro-ISM, Inlab Micro, Inlab Micro Pro-ISM, Inlab Semi–Micro, and Inlab NMR. The electrodes differ in their membrane glass, size, junction and temperature probe, and the sample size and type vary.

Surface sensor

Sometimes, some samples are just too small for even micro sensors to handle accurately. In these situations, surface sensors provide an ideal solution.

Mettler Toledo’s Inlab Surface-Pro-ISM features flat sensing glass membranes paired with a ceramic ring junction. Thanks to the ceramic ring junction, the reference electrolyte can escape and come into contact with the sample. This setup ensures accurate measurements while preventing any contamination of the sample from the reference electrolyte.

The sensor also features a built-in temperature probe and low-temperature glass, making it capable of detecting low ionic concentrations.

Selecting the Right Sensor

As the first step, choosing the right sensor becomes crucial. To do so, consider these important questions:

• The maximum size of the sample: Determine how much sample you can use for accurate measurement.

• Temperatures match: Check if the sample, buffer, and probes can all be at the same temperature, or if you need a temperature probe in the sensor.

• Dirty sample or with particles: If the sample contains dirt or particles, you may need a sensor with an open junction.

• The shape of the container and tube diameter: Consider how the sensor will fit into the container and if it will be fully immersed in the solution.

To make your decision easier, refer to Table 1 for guidance on choosing the right sensor for your needs.

The second step involves calibration. Calibrate the sensor with at least two-point calibration and make sure that the slope is in the range of 95 to 105 per cent and offset between 0 and 20 mV. If it is outside this range, the sensor might need cleaning; if the slope is below 89 per cent, the probe needs to be replaced. Clean the electrode between each buffer.

After the calibration, measure the sample and make sure that the probe membrane and junction are fully covered in the sample. For the micro electrodes the container should not too large or too small. While using Inlab Surface Pro-ISM, place a droplet on a clean and dry surface and place the sensor on top of it making sure that the sample spreads across the membrane and junction surface. Clean the electrode between each sample.

To learn more about how to measure pH in small-volume samples, you can also download an application note.

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