pH measurement with a spectrophotometer

What is it about?

Typically, pH is measured with a pH meter, however, for crystallisation plates solutions this is impractical. To overcome this, we added the pH indicator, bromothymol blue, to small volume of the crystallisation solution and observed the colour change. Using a spectrophotometer we were able to assign the various colours to known pH values. With this set of values as a standard, we can assign a pH value to a new solution by comparing the colour obtained with those for the standard pH values.

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Why is it important?

X-ray crystallography- the most widely used method for determining protein structures, is dependent on pH. The ability to accurately record it will facilitate the reproducibility of experiments and allow for better data-mining studies. Measuring pH with a meter is not only extremely time consuming, but certain chemicals can have a tendency to damage the probe. The spectrophotometric method described here allows the pH to be determined quickly and easily.