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.
