pH is a measure of the hydrogen ion concentration in solution. Most biochemical and chemical reactions are pH-sensitive, making accurate pH measurement and buffer preparation a prerequisite for reproducible results.
The pH Electrode
The standard pH electrode is a combination electrode containing a glass membrane that develops a potential proportional to the hydrogen ion activity. This potential is measured against an internal reference electrode. The meter converts the voltage to a pH reading.
For accurate measurement, calibrate the electrode daily with at least two buffers (typically pH 4.0, 7.0, and 10.0). The calibration slope should be between 95–102% of the theoretical Nernstian slope (59.16 mV/pH at 25 °C).
Electrode Care
Store electrodes in storage solution or pH 4 buffer — never in distilled water, which leaches ions from the glass membrane. If the response becomes sluggish, clean the electrode with 0.1 M HCl followed by 0.1 M NaOH, then rinse and recalibrate.
When measuring, stir the solution gently and allow the reading to stabilize. Temperature affects both the electrode response and the buffer equilibrium — always measure at the same temperature as the calibration buffers or use a meter with automatic temperature compensation.
Buffer Theory
A buffer resists pH change by containing a weak acid and its conjugate base. The Henderson–Hasselbalch equation describes the relationship:
pH = pKa + log([A⁻]/[HA])
The buffer is most effective within ±1 pH unit of its pKa. For maximum capacity, the concentrations of the acid and conjugate base should be roughly equal.
Common Laboratory Buffers
- Phosphate (pKa₂ ≈ 7.2): widely used for biological assays but can precipitate with divalent cations.
- Tris (pKa ≈ 8.07 at 25 °C): common in molecular biology; its pH is strongly temperature-dependent (ΔpKa/°C ≈ −0.028).
- HEPES (pKa ≈ 7.55): a Good buffer with minimal metal binding, ideal for cell culture.
- Acetate (pKa ≈ 4.76): used for DNA extraction and chromatography.
- Citrate (pKa ≈ 3.13, 4.76, 6.40): useful over a broad acidic range.
Preparing a Buffer
Weigh the calculated mass of the buffering agent, dissolve in about 80% of the final volume of distilled water, adjust the pH with HCl or NaOH while stirring, then bring to the final volume. Always adjust the pH at the working temperature, and confirm the pH after bringing to volume.
