Equilibrium between the Stationary and Mobile Phases...

As previously discussed, our sample exists in equilibrium between the mobile and stationary phases. This equilibrium is a random process. Some particles will remain in the stationary phase longer than they will remain in the mobile phase, and vice versa. However, as with our dice rolling example, this process is described by a Gaussian distribution and there is an easily discernable mean time that a particular sample component will linger in the stationary phase before it is eluted. This is known as the retention time, and is measured at the peak of a chromatogram (t_R).

Not all species will be retained by a stationary phase (for instance the mobile phase will not be retained) and will be eluted at the rate of flow of the mobile phase. This mobile phase time (t_M) is used for normalization when comparing retention times between peaks.

 

Not only does a chromatogram detail the times that eluents come off the column, but it the detector will signal how much analyte is eluted at a particular time. By integrating under these curves to find the total area, we can obtain the relative concentration of analyte. This is accomplished in a variety of ways. Mechanically, one could weigh the actual plot paper underneath a curve and then compare this to the weight of paper found under a chromatograph curve of a known volume of analyte. Graphically, the integration could be calculated by hand, or more accurately, by computer methods.