Accurate determination of pH is important due to its effect on enzyme activity and other biological and chemical applications. However, data collection using electrodes is not compatible with high throughput approaches and low volumes inherent to microplate experiments. Here we describe a spectrometer-based method using the SPECTROstar® Nano microplate reader. The phenol-based dye bromothymol blue is ideally suited for colorimetric assays to study pH in the physiological range. This rapid method accurately measures pH and can be easily placed in line with Hudson’s SOLO Automated Pipettor and Micro 10X Dispenser for plate preparation. Complete automation can be attained by including a robotic arm such as the PlateCrane EX from Hudson.
Using either the basic or the neutral form of bromothymol blue provides equivalent prediction of the pH of unknown samples. The absorbance spectra obtained on the SPECTROstar Nano can be easily analyzed with the MARS data analysis software to re-calculate the pH-value of unknown samples.