Segmented milli-fluidic crystallisation of paracetamol with <i>in situ</i> single-crystal X-ray diffraction

Serial crystallography has revolutionalised our ability to analyse protein crystals; crystal structures can be uncovered by combining data from multiple crystals mitigating radiation damage through overexposure to the X-ray beam. With synchrotron sources becoming even brighter, radiation damage is ever more pertinent for small molecule crystals as well. Combining serial crystallography with flow crystallisation, we pave the way for exploring high-throughput screening and kinetic studies, with application to small molecule crystals up to mm-scale. Here we present the first known example of single crystal X-ray diffraction of small molecule crystals in a flow crystallisation environment. <i>In situ</i> single-crystal X-ray diffraction has been achieved on a series of growing singular crystals through the use of segmented flow cooling crystallisation, holding crystals in an X-ray beam for 4.2 s whilst they freely rotate whilst flowing inside tubing. Upon triggering of a passing slug at the analysis point, the tubing is moved in the opposite direction to the flow, enabling the crystal to remain within the X-ray beam whilst maintaining the free rotation of the crystal due to fluid movement. Structure solution of paracetamol form I has been achieved with 0.8 Å resolution using data combined from 13 crystals whilst unit cell information can be extracted from a single crystal.