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			96-Well Plate

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	48-602MAG	Buffer Detection Kit for Magnetic Beads	1 Kit

Water for pH Measurement

Impact of Water

Buffers and solutions for pH measurement are prepared from solid reagents that are dissolved in water, or from pre-made concentrated solutions that can be diluted with water. The water used to prepare these solutions should be pure, devoid of significant concentrations of acids or bases, and free of substances that can modify the pH of the solutions.

Water Quality Parameters

Ions
Significant contamination by ions such as ammonium and carbonate can alter the hydrogen ion concentration of a solution, resulting to inaccurate pH measurements. When preparing buffers of specific ionic strength, the presence of all ions at high concentration will modify the ionic strength of the solutions. It is therefore safe to select water with low ionic concentration; a resistivity > 1 MΩ.cm is recommended.

Organics
Water that that is heavily contaminated with weak organic acids or bases could modify the pH of a solution. In order to ensure low concentration of those organic molecules, the overall concentration of organic in the water should be reduced. Water with a fairly low TOC (below 50 ppb) should be selected.

Bacteria
While bacteria would not withstand strong acids or bases, they may develop in buffers at pH around 5 to 9. In addition, bacteria release charged organics upon when their membrane disrupts. Water with low bacterial content would avoid any problem linked to bacteria and their by-products.

Particles
Although not very sensitive to particles, the performance of a pH meter could be affected by high concentration of particles, which could aggregate on the surface of the electrodes. A filtration step at the end of the water purification process is recommended.

Water Storage
While this is not a water quality parameter stricto senso, water storage can have a dramatic effect on the final pH of solutions, in particular when pure water is stored before preparing the solutions. After purification water is very pure and ions and gases can dissolve readily. Upon contact with air, CO2 from air dissolves in water to generate bicarbonate and carbonate, essentially turning the stored pure water into a carbonate buffer. Mixing that carbonate solution to pre-made solutions or with powders is likely to result in a pH different than the expected.

In addition, bacteria can develop readily in stored water, and they will create biofilms in the container typically used to store the water and solutions/buffers. It is therefore highly recommended to clean thoroughly the bottles used to prepare and keep the buffers between each use.

Experimental results that illustrate the use of high purity water for pH measurements

An experiment was designed to demonstrate that pure water delivered by water systems combining reverse osmosis and electrodeionization (Elix®) and ultrapure water from a polishing unit (Milli-Q®) could be used to prepare buffer solutions. Phosphate and PBS stock solutions were diluted 10- and 100-fold. Results show that the pure and ultrapure water freshly produced give identical results to bottled distilled water for buffer preparations. Water purification systems provide the benefits of having pure water on demand, which can also be used for other applications in the lab, and a reduced cost of pure water per liter.

Merck:/Freestyle/LW-Lab-Water/applications/pH/LW-LC-pH-Image1-460x207.jpg

Figure 1. Effect of dilution of stock solutions using different sources of water. Milli-Q® water: resistivity 18.2 MΩ.cm, TOC < 5 ppb, bacteria 1 < cfu/mL. Elix® water: resistivity > 5 MΩ.cm, TOC < 30 ppb (measured in-line) and bacteria < 10 cfu/mL.

It should be remembered that temperature also has an impact on the pH value. Preparing buffers with very cold solutions may results in differences in pH of 0.1.

The pH of Milli-Q® Water

The pH of Milli-Q® water is equal to 6.998, as demonstrated in the article “High Purity Water and pH” (see link below).

However, as Milli-Q® water is very pure, it contains very few ions and has a low buffering capacity. Once water has been sourced from the system, its pH value can therefore change quickly. Any impurity on the wall of the container used to collect the Milli-Q® water will dissolve and change the pH value; carbon dioxide naturally present in the air will dissolve in the water, producing bicarbonate and decreasing the pH value.

In addition, regular pH meters available in most laboratories have not been designed to operate in a solution containing almost no ions. They are not adapted to measure the pH of Milli-Q® water and may therefore deliver inaccurate results; some pH meter suppliers provide documents explaining the technical reasons why conventional pH meters do not provide accurate results when measuring the pH of ultrapure water. Some companies have designed pH meters able to measure the pH of ultrapure water in dynamic operating conditions, but these are rarely used in regular research laboratories.

This is the reason why scientific forums regularly report Milli-Q® water pH measurements far above 7.0, even though the pH of Milli-Q® water leaving the purification system is actually very close to 7.

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