In-line spectroscopic measurement of pH profiles using methyl orange: Application to pH transients in protein A...

In this work, we developed a method using the pH indicator methyl orange and an in-line UV detector to obtain accurate pH profiles for chromatographic applications in the pH range from 3.2-4.5. A linear calibration was used for conversion of the ratio of absorbances at two wavelengths to pH and the method was shown to be relatively insensitive to ionic strength and buffer composition over the range of conditions examined. Methyl orange pH profiles were then validated against both offline and in-line pH probes, as well as a buffer mixing model. The approach was extended to measurements with a protein A column using a small mixer downstream of the column to enable addition of methyl orange prior to UV detection while avoiding column exposure. This approach was then employed in concert with in-line conductivity measurements to examine pH transient effects in a variety of buffer systems, revealing a wide range of pH transient behavior. The rapid response time of methyl orange compared to conventional pH electrodes was shown to yield improved accuracy under certain conditions and the observed data shed light on pH transitions of interest in affinity chromatography.