Instrumental Idiosyncrasies Affecting the Performance of Ultrafast Chiral and Achiral Sub/Supercritical Fluid Chromatography

It
is widely accepted that column technology is ahead of existing
chromatographic instruments. The chromatographic output may not reflect
the true picture of the peak profile inside the column. The instrumental
optimization parameters become far more important when peaks elute
in a few seconds. In this work, the low viscosity advantage of the
supercritical/subcritical CO2 is coupled with the high
efficiency of narrow particle size distribution silica. Using short
efficient columns and high flow rates (up to 19 mL/min), separations
on the order of a few seconds are demonstrated. In the domain of ultrafast
supercritical fluid chromatography (SFC), unexpected results are seen
which are absent in ultrafast liquid chromatography. These effects
arise due to the compressible nature of the mobile phase and detector
idiosyncrasies to eliminate back-pressure regulator noise. We demonstrate
unusual connection tubing effects with 50, 75, 127, 254, and 500 μm
tubings and show the complex relation of dead time, retention time,
efficiency, and optimum velocity with the tubing diameter (via column
outlet pressure). Fourier analysis at different back-pressure regulator
(BPR) settings shows that some instruments have very specific noise
frequencies originating from the BPR, and those specific frequencies
vanish under certain conditions. The performance of embedded digital
filters, namely, moving average, numerically simulated low pass RC,
and Gaussian kernels, is compared. This work also demonstrates, using
a simple derivative test, that some instruments employ interpolation
techniques while sampling at “true” low frequencies
to avoid picking up high frequency noise. Researchers engaged in ultrafast
chromatography need to be aware of the instrumental nuances and optimization
procedures for achieving ultrafast chiral or achiral separations in
SFC mode.