Various applications in life science research involve small samples, as the substance or active ingredient is only available in low quantities, and the production of the sample often is costly and labor-intensive. As the pH of a solution is directly proportional to the concentration of hydronium ions in the sample, it may not be feasible to obtain an exact pH value by diluting the sample. But the good news is that there are sensors available that can help to measure pH in small samples directly without diluting them. When choosing a sensor, we need to think about factors that affect the pH measurements, such as the sensing glass, junction, temperature and sample. For small samples, the size and shape of the container should also be considered.

Micro sensors

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.

InLab Ultra Micro-ISM has low-temperature glass that allows it to be measured at low temperatures and low ion concentrations. Furthermore, this sensor has a ceramic junction that allows electrolytes to flow out slowly and is perfect for aqueous solutions; this junction is not optimal for samples with high viscosity or alkalinity above pH 11. This sensor has a 3.0 mm shaft diameter that allows it to measure pH in 384 microwell plates and only needs 15 μl, and in 96 well plates and 1.5ml tube, 20-25 μl would be needed. But for this sensor, it is better to keep the sample temperature constant as it doesn’t have a temperature probe.

InLab Micro and InLab NMR also have narrow shafts of 3 mm but different shaft lengths of 60 mm and 200 mm; both have ceramic junctions and allow measurement in 96 well plates with the lowest volume of 45 μl. These sensors have a universal glass membrane that allows measuring in the full pH range of 1-14. InLab NMR with a long shaft is perfect for measuring in NMR tubes. These probes don’t have a temperature probe, so micro sensors and samples should be stored at the same temperature to get accurate results and avoid errors due to temperature changes.

If the user will not be able to control sample temperature, then the sensor to choose is the InLab Micro Pro-ISM. This sensor has a built-in temperature probe, a wide measurement range with a universal glass membrane, and a ceramic junction. The sensor shaft is 5 mm wide, and as it is slightly larger than the other sensors, the minimum volume is 100 μl, but depending on the container, it might need up to 500 μl.

For a dirty sample, a researcher might consider InLab Semi–Micro, which has an open junction that doesn’t clog easily and has Xerolyt Extra polymer soaked in electrolyte. Also, A41 glass is resistant to chemicals. With a 6 mm wide shaft, the minimum sample volume is 100 μl, but the container matters.

Surface sensor

If you have a limited amount of sample, just a droplet, the InLab Surface-Pro-ISM is an excellent option to consider. This electrode uses flat sensing glass membranes and a ceramic ring junction, which ensures accurate and reliable measurements. Additionally, it features a built-in temperature probe and low-temperature glass, making it capable of detecting low ionic concentrations.

Flex sensor

If dealing with small sample volumes with higher protein content, the InLab Flex-Micro is a flexible sensor and could be a great solution. While it requires a minimum volume of 500 μl, this probe is mechanically robust. Thanks to its epoxy construction, it is easy to handle and less prone to breaking. Moreover, it features a PTFE junction with pores that have a lower chance of getting clogged.

Source: Mettler ToledoSource: Mettler Toledo

How do we measure small samples with micro, surface and flex electrodes?

Step 1: Choose the right sensor for an application. Questions to ask are: What is the maximum sample that can be used for measurement? Can the temperature of the sample, buffer and probes be kept the same, or do is a temperature probe needed in the sensor? Is the sample dirty, suspended with particles, and would an open junction be needed? What is the diameter of the tubes and container shape, will the sensor be immersed in a solution with the container available? To sum it up, let's have a look at Table 1 to choose the right sensor.

Table 1. Source: Mettler ToledoTable 1. Source: Mettler Toledo

Step 2: Calibrate the sensor with at least two-point calibration and make sure that the slope is in the range of 95% to 105% and offset between 0 and 20 mV. If it is outside this range, the sensor might need cleaning; if the slope is below 89%, the probe needs to be replaced. Clean the electrode between each buffer.

Step 3: Measure the sample and make sure that the probe membrane and junction are fully covered in the sample. For the micro electrodes make sure that the container is 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 about how to measure pH in small-volume samples, download this application note.


To contact the author of this article, email pHmatters@mt.com