The bottleneck of sample analysis has always been the sample preparation. Online SFE-SFC-MS/MS minimizes
sample preparation by allowing solid samples
to be completely analyzed without performing
sample extraction and cleanup at the lab
bench. The online method allows automatic
solid sample extraction to be completed. This
is subsequently followed by chromatography
separation and ultimately detection by triple
quadrupole mass spectrometry. The online
extraction process is up to seven times faster
than the current QuEChERS method for
pesticide analysis in food. Other fast sample
preparation applications include preserving
labile compounds in dietary supplements,
cleaning validation in pharmaceuticals,
biomarkers for dried blood spots, and trace
additives in polymers.
Supercritical fluids (SCF) have some
properties similar to liquids and others
similar to gases. For example, SCF density
behaves more like a liquid due to its higher
density and therefore higher solubility.
With respect to viscosity and diffusivity,
SCF behaves more like a gas. The higher
diffusivity provides faster mixing and better
extraction. The lower viscosity allows lower
pressure, higher flow rates and faster analysis
times, similar to a gas chromatograph (GC).
SFC with added high pressure (9600 psi or
66MPa) can reduce analysis time by two-thirds that of HPLC methods.
The “Achilles Heel” of the SFC-MS/MS
interface has been the back pressure regulator
(BPR). The purpose of the BPR is to keep
the SCF in that state through pressure. The
problem is current technologies require
splitting the sample 4:1 between the BPR and
the MS to avoid peak broadening in the BPR.
As a result, only 20 percent of the sample
BPR splitless design. The new SFC-MS/MS
technology enhances sensitivity by at least five
times compared with current technologies for
SFC-MS/MS, as shown in Figure 1.
The first supercritical fluid
chromatography (SFC) reference is credited
to Klesper et al. in 1962. The authors
described the technique as high-pressure
gas chromatography (HPGC). In their
work, the supercritical fluids used were
dichlorodifluoromethane (Tc = 112°C, Pc =
1000 atm) and monochlorodifluoromethane
(Tc = 96 °C, Pc = 1400 atm).
In the mid-1960s, Sie[ 2-6] et al. published
a series of papers using carbon dioxide (Tc =
31.3°C, Pc = 72.9 atm) as the mobile phase.
Since carbon dioxide has a low supercritical
temperature and pressure, it has been used
extensively since the 1980s as an extraction
method. CO2 offers a number of other
advantages, including being inexpensive, nontoxic, non-flammable and relatively “green”
compared to other solvents with respect to
The first coupling of SFE to SFC with a
capillary column was by Hawthorne [ 7] and
Wright [ 8] in 1987, while McNally[ 9] and
Engelhardt[ 10] reported the coupling with a
packed column in 1988. Packed column is the
choice of methods with SFC today.
This article introduces the analysis of
solid samples with little sample preparation
in the areas of foods, dietary supplements,
pharmaceutical, clinical, and polymers using
the first online SFE-SFC-MS/MS.
Shimadzu’s Nexera UC (Unified
Chromatography) was used for all
experiments. The online configuration
was SFE-SFC-MS/MS. The Nexera UC
system can also use other detectors such as
UV-Vis, Photodiode Array (PDA), and single
quadrupole mass spectrometers.
Results and Discussion
Sample preparation may involve many
steps such as blending, shaking, filtering,
partitioning, centrifuging, evaporating,
concentrating, extraction, and cleanup as well
as using lots of glassware and solvents. The
ideal workflow would allow analysis of solid
samples with minimal sample preparation
and without human intervention. This ideal
workflow is accomplished with online SFE-SFC-MS/MS or SFE-SFC-UV.
Solving Sample Prep Bottleneck
Across Multiple Industries
Online SFE-SFC-MS/MS can help minimize sample prep, thus speeding time
to results and overall workflow.
Figure 1: Sensitivity Comparison of Split vs. Splitless SFC-MS/MS Designs.