HPLC is a modern separation technique used for complex samples. It is used in a wide variety of fields such as toxicology, environmental chemistry, and the pharmaceutical industry. The purpose of this research project was to examine the effect extra column volume (ECV) has on a chromatographic separation. This was studied using varying lengths of connecting tubing from the injector to the column and from the column to the detector. Learning more about the effects of ECV on the separation achieved is beneficial to scientists that are currently using HPLC systems and wish to improve the efficiency of sample analysis without the significant cost of new systems or having to go through method development and validation. It is known that excess connecting tubing can significantly affect band broadening, so adjusting the tubing length could be an effective and inexpensive solution for these scientists. The experimental procedure was performed using two Perkin Elmer 200 Series HPLC systems including pump, interface, and detector. Each system utilized a Rheodyne 7725i manual injector. The experiment required using PEEK tubing with differing internal diameter. The three sizes used were 0.005, 0.010, and 0.20 inch internal diameter. An ODS, 150 x 4.6 mm column with 5 micrometer particle size was used and a systematic study was performed to determine the effects of different lengths of connecting tubing on the variance associated with the peaks.