Graduate students play a critical role in undergraduate education at doctorate granting institutions; but generally have minimal opportunity to develop teaching expertise. Furthermore, little is known about how graduate students develop teaching expertise in this context. We investigated the development of topic-specific pedagogical content knowledge among chemistry graduate student teaching assistants (GTAs). Thin layer chromatography was selected as the topic of investigation because undergraduate students encounter it throughout organic chemistry lab and it is connected to several foundational chemistry topics. An instrument was developed to measure both content knowledge (CK) and pedagogical content knowledge (PCK) of thin layer chromatography for GTAs with a range of teaching experience. Data from the test instrument were transformed using the Rasch model and statistically analysed. Our analysis showed that graduate students at all levels of experience performed well on content knowledge questions, but even experienced GTAs demonstrated low levels of pedagogical content knowledge. Importantly, experienced GTAs demonstrated a greater proficiency than novice GTAs, which suggests that pedagogical content knowledge is developed over time.