All chromatographic systems have a mobile phase that transports the analytes through the column and a stationary phase coated onto the column or on the resin beads in the column. The stationary phase loosely interacts with each analyte based on its chemical structure, resulting in the separation of each analyte as a function of time spent in the separation column. The less analytes interact with the stationary phase, the faster they are transported through the system. The reverse is true for less mobile analytes that have stronger interactions. Thus, the many analytes in a sample are identified by retention time in the system for a given set of conditions. In GC, these conditions include the gas (mobile phase) pressure, flow rate, linear velocity, and temperature of the separation column. In HPLC, the mobile phase (liquid) pressure, flow rate, linear velocity, and the polarity of the mobile phase all affect a compounds’ retention time. An illustration of retention time is shown in Figure 1.2. The equation at the top of the figure will be discussed later during our mathematic development of chromatography theory.
Figure 1.2. Identification of Analytes by Retention Time.
In the above figure, the minimum time that a non-retained chemical species will remain in the system is tM. All compounds will reside in the injector, column, and detector for at least this long. Any affinity for the stationary phase results in the compound being retained in the column causing it to elute from the column at a time greater than tM. This is represented by the two larger peaks that appear to the right in Figure 1.2, with retention times tRA and tRB. Compound B has more affinity for the stationary phase than compound A because it exited the column last. A net retention (tR’A and tR’B) time can be calculated by subtracting the retention time of the mobile phase(tM) from the peaks retention time (tRA and tRB).
Figure 1.2 also illustrates how peak shape is related to retention time. The widening of peak B is caused by longitudinal diffusion (diffusion of the analyte as the peak moves down the length of the column). This relationship is usually the reason why integration by area, and not height, is utilized. However, compounds eluting at similar retention times will have near identical peak shapes and widths.
A summary of these concepts and data handling techniques is shown in Animation 1.1. Click on the figure to start the animation.