Post column derivatisation analyses review. Is post-column derivatisation incompatible with modern HPLC columns?
Post Column derivatisation (PCD) coupled with high performance liquid chromatography or ultra-high performance liquid chromatography is a powerful tool in the modern analytical laboratory, or at least it should be. One drawback with PCD techniques is the extra post-column dead volume due to reaction coils used to enable adequate reaction time and the mixing of reagents which causes peak broadening, hence a loss of separation power. This loss of efficiency is counter-productive to modern HPLC technologies, -such as UHPLC. We reviewed 87 PCD methods published from 2009 to 2014. We restricted our review to methods published between 2009 and 2014, because we were interested in the uptake of PCD methods in UHPLC environments. Our review focused on a range of system parameters including: column dimensions, stationary phase and particle size, as well as the geometry of the reaction loop. The most commonly used column in the methods investigated was not in fact a modern UHPLC version with sub-2-micron, (or even sub-3-micron) particles, but rather, work-house columns, such as, 250 × 4.6 mm i.d. columns packed with 5 μm C18 particles. Reaction loops were varied, even within the same type of analysis, but the majority of methods employed loop systems with volumes greater than 500 μL. A second part of this review illustrated briefly the effect of dead volume on column performance. The experiment evaluated the change in resolution and separation efficiency of some weak to moderately retained solutes on a 250 × 4.6 mm i.d. column packed with 5 μm particles. The data showed that reaction loops beyond 100 μL resulted in a very serious loss of performance. Our study concluded that practitioners of PCD methods largely avoid the use of UHPLC-type column formats, so yes, very much, PCD is incompatible with the modern HPLC column.
Australian Centre for Research on Separation Sciences (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), NSW 2150, Australia.
Andrew
A
Jones
Australian Centre for Research on Separation Sciences (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), NSW 2150, Australia.
Sercan
S
Pravadali-Cekic
Australian Centre for Research on Separation Sciences (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), NSW 2150, Australia.
Gary R
GR
Dennis
Australian Centre for Research on Separation Sciences (ACROSS), School of Science and Health, University of Western Sydney (Parramatta), NSW 2150, Australia. Electronic address: r.shalliker@uws.edu.au.
R Andrew
RA
Shalliker
Amino Acids; Antioxidants; Chromatography; HPLC; Post column derivatisation