In HIONP Hexane is a non-polar compound and it has a neutral pH so the sample is completely acidic due to the negatively charged Nigella sativa and also the drying could have also impacted the pH, therefore, the value is -11.8mV. Thymoquinone though neutral but because of its instability and light sensitivity and traces of impurities during HPLC extraction could have caused fluctuations in pH and reduced the zeta potential drastically leading to instability TQ IONP. The reason for adding DMSO is that it dissolves thymoquinone and it is highly alkaline which could counteract the negatively charged Nigella sativa and increase stability. The aim of the current research was to prepare stable aggregates of Thymoquinone iron oxide nanoparticles, this research can be further extended to test for their antibacterial activity on various gram-negative species of bacteria.