An optimization of a cleaning in place (CIP) protocol has been performed for STREAMLINE Q XL separation medium using homogenised Escherichia coli as sample. The effectiveness of the different cleaning protocols was tested by analysing the content of fatty acids, phosphorus compounds and amino acids in STREAMLINE Q XL. Additionally, RAMAN and FT-IR measurements were used to verify the degree of contamination of separation medium. From the results of the optimization experiments, a cleaning procedure was suggested and subsequently tested with 50 repetitive applications. The study shows that of the different agents tested, 1 M NaOH is the most efficient cleaning solution and that long contact time (~8 h) with 1 M NaOH is important. An additional cleaning step with 30% 2-propanol (v/v) is also recommended for E. coli samples.