Translate this blog

Sunday, February 3, 2013

How Analyte Loading Affects Peak Shape & Method Development


Different modes of chromatography require different thinking. In reversed phase (RP), the amount of analyte you can load onto the column without affecting retention or peak shape is not necessarily the same as with Aqueous Normal Phase (ANP) mode. Analytes in reversed phase solvents have different solubilities than in ANP and the retention mechanisms between the two are different as well.

                A given vitamin analyte for example may be retained in both RP and ANP, but the concentration and/or injection volume limits of each mode should be considered separately. An injection volume of 20 µL may be fine for a particular RP method but the same injection volume could produce a tailing peak in ANP under certain conditions. The concentration should also be considered, since the key aspect is the total amount of analyte in a given injection plug. An analyst that observes the tailing peak may incorrectly attribute it to silanol interactions and spend unnecessary amounts of time investigating other mobile phase conditions to reduce the tailing. In this case, understanding the different load limits of the column can make method development faster and easier by anticipating problems before they are observed.

                A study was done on a water soluble vitamin in ANP mode in which a series of runs was performed and only the injection volume was varied with each method. Under these conditions, it was observed that while an injection volume of 1 µL produced a symmetrical sharp peak, progressively increased tailing and decreased retention were observed with higher injection volumes. The 10 µL peak was significantly lower in retention and produced unacceptable tailing.

 

No comments:

Post a Comment