Getting Started in HPLC

Section 1D. Chromatography by the Numbers.

   
In the original use of chromatography, a band could be identified by the distance traveled in a constant time. In the example at the right, the red pigment travels further than the blue pigment which travels further than the green pigment. If the separation is repeated under identical conditions (not always easy to do!), each of the pigments will always travel its characteristic distance, determined by the balance between solubility in the mobile phase and affinity for the stationary phase.


   
As we have seen, a chromatogram is a plot of detector response as a function of time. In a properly functioning HPLC system, the detector response is directly proportional to the concentration of analyte in the column effluent. At constant flow rate, the volume of column effluent that has passed through the detector is directly proportional to the elapsed time.

In HPLC, all analytes travel the same distance (the length of the column), so we characterize compounds by retention time. Faster-moving analytes have a shorter retention time than slower-moving analytes. Retention time, like migration distance in the original chromatography experiment, is determined by the balance between solubility in the mobile phase and affinity for the stationary phase. If the separation were repeated under identical conditions (much easier to do in HPLC!), then each analyte would always elute from the column at its characteristic retention time. That retention time can be used to identify compounds (qualitative analysis).

 

 
   
By convention, the retention time (tR) is measured from sample injection (the start of the chromatogram) to the apex (high point) of the peak. Retention times are usually determined automatically by the data system. They are often included on the chromatogram printout as well as being available in a report about the separation.


Chromatogram with 9 peaks showing retention times.


   
If standards (sometimes also called "calibrators") containing known compounds are run, then the data system can "remember" the retention times and automatically associate analyte names with the peaks. The example at the right shows a data system report that has both peak names and retention times.


Data system report showing peak retention times, area, height, and names. The names are established by running standards.


   
 

 


2000, LC Resources Inc. All rights reserved.
Last revised: March 30, 2001.