The purpose of this project was to investigate if retention of polar compounds that are given to treat tuberculosis, diabetes, inflammatory bowel disease and childhood leukemia could be obtained with HILIC separation. By varying different parameters for different types of columns the compounds were analyzed with the aim of finding guidelines for future method optimizations. To perform these analyzes three different columns were tested – ZIC-HILIC (silica-based with zwitterions), ZIC-pHILIC (polymer-based with zwitterions) and XBridge Amide (amide functions). The results were evaluated with selected quality measures. The parameters being varied were pH, temperature, flow rate, type of buffer and ionic strength. In addition, comparisons between isocratic and gradient separations were performed.   Over 1 000 analyzes were conducted in which retention of 16 of total 18 substances were successfully obtained with HILIC. The columns that generated the best results in terms of greatest number of identified compounds were ZIC-HILIC and XBridge Amide. ZIC-pHILIC offered a wide pH range but generally gave inferior chromatography. The influence of the different parameters on the results has not been investigated in sufficient scope. This means that no specific methods for the different drug groups can be reported.