pH Measurement of Small-Volume Samples

Streamline Small Sample pH Measurements through Automation

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Measuring the pH of small, precious samples is a recurring challenge in pharmaceutical and biopharmaceutical laboratories. Conventional pH electrodes often struggle in such scenarios, where limited volumes restrict sample interaction and complicate sensor insertion. To overcome these hurdles, METTLER TOLEDO’s InLab Micro Pro-ISM and other specialized pH sensors are designed to deliver precise, reproducible measurements, even with microliter-scale volumes.

This application note explains how the InLab Micro Pro-ISM sensor supports small-volume pH testing, highlights good electrochemistry practices, and shares experimental data demonstrating reliable results across biologically relevant solutions.

Figure 1. Accurate results with METTLER TOLEDO’s InLab® Micro Pro-ISM sensor.

The InLab Micro Pro-ISM is engineered specifically for small-volume pH testing. Its narrow shaft and advanced design ensure accurate measurements in tight containers while maintaining long-term stability.

Key features include

5 mm glass shaft — allows effortless insertion into narrow containers, enabling measurement of limited, high-value samples.
Integrated temperature probe — ensures correct sample temperature capture, making setup leaner and more accurate.
Ceramic junction — supports optimal electrolyte flow in aqueous solutions for accurate, stable results; highly durable and easy to maintain.
ARGENTHAL™ reference system — prevents silver ion leaching into the sample, avoiding precipitation and junction contamination that can destabilize readings.
Intelligent Sensor Management (ISM®) — provides traceability by tracking calibration data, temperature exposure, and sensor condition for secure data management.

Accurate small-volume pH measurement requires not only the right sensor but also correct calibration, sample preparation, and maintenance practices. Table 1. Features and advantages of InLab® Micro Pro-ISM sensors.

Table 1. Features and advantages of InLab® Micro Pro-ISM sensors.

Calibrate sensors with METTLER TOLEDO buffer solutions before the first sample of the day. For >30 samples/day, recalibrate halfway through.
Use at least two buffer standards around the expected pH range (e.g., pH 7.00 and 9.21 for a sample near pH 8.19).
Ensure calibration slope falls between 95–105% and zero offset is within ±20 mV at pH 7.
Verify accuracy with an additional buffer (e.g., pH 8.00 for the above case).
Recheck after every 10 samples; recalibrate if necessary.
Always use fresh buffers and ensure measurement conditions (temperature) are consistent.

Samples tested: phosphate-buffered saline (PBS), Bovine Serum Albumin (BSA) in PBS, and DNA (sodium salt from salmon testes) in tris buffer.
PBS buffer was used as received.
BSA prepared at 1% (w/v) in PBS.
DNA prepared at 250 mg/100 mL in tris buffer (pH 8).
pH measured in automatic end-point mode, triplicate per sample.
After each run: clean electrode with deionized water, dry gently with lint-free tissue to avoid carryover.

For protein-rich samples, soak in pepsin/HCl solution (5% in 0.1 M HCl) for ≥1 hour after use to prevent junction clogging.
Periodically recondition in 0.1 M HCl depending on usage and sensor age.
Always recalibrate after reconditioning.
Store in wetting cap with InLab® Storage Solution; never dry-store or use distilled water.

Using the InLab Micro Pro-ISM, three samples (PBS, BSA in PBS, DNA in tris buffer) were measured with volumes as low as 150 μL in 500 μL vials.

All results showed excellent reproducibility with standard deviation <0.05 pH units.
Average response times were under 20 seconds.
Using the InLab Surface Pro-ISM with an InMotion™ Autosampler, reliable pH measurement was also demonstrated on 1 mL samples, maintaining precision below 0.05 pH units.

Figure 3. pH Measurement of small volume PBS sample in a 500 μL vial.

Sensor	Sample	pH	Std. Dev.	Res. Time (s)	Temp. (ºC)
InLab® Micro Pro-ISM	PBS	7.30	0.009	09	25.4
	BSA	7.21	0.004	18	25.6
	DNA	8.19	0.002	07	21.9

Table 2. pH measurement of different sample solutions using InLab Micro Pro-ISM sensors.

Beyond the InLab Micro Pro-ISM, METTLER TOLEDO offers several other electrodes optimized for specific small-volume scenarios:

InLab Ultra-Micro ISM (3 mm shaft) — enables ultra-low volume testing, including 384-well plates.
InLab Semi-Micro (6 mm shaft) — maintenance-free with open junction, suited for dense samples prone to clogging.
InLab Flex-Micro (6 mm shaft) — robust epoxy shaft design, maintenance-free.
InLab Surface Pro-ISM (12 mm shaft) — flat membrane with built-in temperature probe; ideal for droplet surface pH and automation down to 1 mL volumes.

When combined with InMotion Autosampler and LabX™ software, this sensor enable automated, traceable workflows with centralized data handling.

The InLab Micro Pro-ISM demonstrated high reliability for small-volume pH measurements, consistently delivering precision below 0.05 pH units. It is therefore an excellent choice for laboratories handling limited or precious pharmaceutical and biopharmaceutical samples.

Alternative InLab sensors extend measurement capabilities across even smaller volumes, dense solutions, or droplet surfaces, while automation with InMotion™ further enhances throughput and data management.

By pairing the right sensor with good electrochemistry practices, laboratories can achieve reproducible and efficient pH measurement, even when every microliter counts.

Watch Small-Volume pH Automation in Action

See how METTLER TOLEDO’s automation solutions with InLab Surface Pro-ISM sensor make small-sample pH testing faster and more reliable. With as little as 1 mL of sample, the system automates measurement, sensor cleaning, and even lid handling, saving time while ensuring precise, reproducible results.

Dr. Sagar Patil is an R&D Test Scientist in the pH R&D laboratory at METTLER TOLEDO, where he advances pH measurement technologies and develops innovative solutions for laboratory workflows.

With a Ph.D. in Chemistry and post-doctoral research experience, he applies his expertise in chemical synthesis and instrumentation to create application notes, design products, and ensure precise, reproducible results.