On-Line Supercritical Fluid Extraction/Chromatography System

for Trace Analysis of Pesticides in Soybean Oil

and Rendered Fats
Balasingam Murugaverl and Kent J. Voorhees*
Department of Chemistry and Geochemistry, Colorado School of Mines, Golden, CO 80401

Abstract. A method for trace analysis incorporating solid phase extraction (extraction and clean-up)
with supercritical fluid chromatography (SFC) for analytes in fats and oils has been developed. Direct
coupling (on-line) of supercritical fluid solid phase extraction with supercritical fluid chromatography
(SFSPEISFC) allows analysis of solutes in fats and oils for which on-line SFElSFC is unsuitable. The
main feature of SFSPEISFC is that extraction and clean-up are accomplished in one step using
unmodified supercritical carbon dioxide. Extraction/clean-up is monitored by a photodiode array
detector which allows fractionation. The operational set-up and potential usefulness of the SFSPE/SFC
system and applications for pesticide analysis of lipid sample matrices are described.

Key words: supercritical fluid extraction/chromatography, pesticide analysis, cryogenic trapping

INTRODUCTION tography (SFSPEISFC) has been proposed as a method for

Detection of pesticides and their metabolites from
complex sample matrices such as animal tissue is an
ongoing concern. Demand for trace analysis has increased
in recent years due to increasing public awareness of
environmental pollution. At present about 750 pesticides,
herbicides, and related compounds require analytical
detection. Only about 200 are detected by G C M S
methods. Due to inherent chemical and/or physical
properties, the remaining 550 are analyzed by single
residue methods.
Coupled (on-line) supercritical fluid extraction (SFE)
with supercritical fluid chromatography (SFC) or gas
chromatography (GC) offers potential for consolidating
various single residue detection methods currently em-
ployed. Each approach provides the versatility of super-
critical fluids with near quantitative transfer of extracted
analytes into the analytical column with minimal sample
handling. On-line SFE/SFC and SFC/GC have been
studied by numerous workers (1-11) for extraction and
analysis of analytes from various sample matrices. Their
effectiveness relies mainly on variable solvating power of
a supercritical fluid (as a function of density) to selectively
extract and isolate discrete fractions from sample matrices.
In general, the method has not been utilized for trace
analysis where the analyte is associated with complex
sample matrices such as fats and oils. Co-extraction of
lipids seems unavoidable and adversely affects separation
and detection of analytes (1,2).
An approach called on-line supercritical fluid solid
phase extraction and capillary supercritical fluid chroma-
trace analysis of analytes from complex sample matrices
such as fats and oils (12). Early SFE/SFC approaches
used a solid phase trap for collection of extract prior to
direct elution from the trap onto an analytical column;
however, no sample fractionation occurred. The SFSPEI-
SFC method differs from on-line SFE/SFC since the solid
phase trap is used for trapping as well as clean-up.
Collection and clean-up are accomplished in one step prior
to introduction of the analyte into the analytical column,
thus allowing lipid-containing sample matrices to be
analyzed. Unmodified carbon dioxide may be used as the
supercritical fluid in the system since selective extraction
is no longer crucial. Unique advantages result over
modified mobile phases, including low critical temperature
and pressure, no waste disposal problems, and more
importantly, the use of a variety of detectors. Instrumen-
tation and application of an on-line SFSPE/capillary SFC
method of analysis for pesticides from lipid sample
matrices are discussed.
EXPERIMENTAL
Samples. Pesticide reference standards (diuron,
alachlor, bendiocarb and carbaryl) were obtained from
EPA-Research Triangle Park,NC. Soybean oil and pork
lard were purchased from a local supermarket. Beef and
bacon fat samples were prepared by rendering beef and
bacon.
Instrumental. The on-line SFSPEISFC instrumenta-
tion consisted of an extraction cell, a packed trapping and
clean-up column, a cryofocusing interface, and a capillary

*Author to whom correspondence should be addressed

* 1991 MicroSeparations
J. Microcol. Sep. 3, 11-16 (1991) 11
12
i co2
hi.
H e a t e d Coil
Port
\ Jen'
Photodiode
Array Detector
' I
d
Packed
Column
1
Capillary Column
Figure 1. Schematic illlistration of the SFSPEISFC set-up.
SFC (Figure 1). Supercritical fluid chromatographic grade
CO, (Scott Specialty Gases) delivered by a Varian 8500 or
a Lee Scientific model 600 syringe pump was used as the
supercritical fluid mobile phase. The mobile phase density
was computer controlled. Packed column blanks were
constructed from 35-mm x 2-mm i.d. stainless steel tubing
and 114 to 1/16-inch stainless steel zero dead volume
Swagelock column end fittings with built-in 2-pm pore size
stainless steel frits. Columns were dry packed with
appropriate sorbents using the tap and fill method.
Sorbents (40-pm average particle diameter and 60-A pore
size) were obtained from Sep-Pak solid phase extraction
tubes. A Hewlett-Packard model 8452 photodiode array
detector with a custom constructed capillary flow cell
holder (11) was used to monitor the extract/clean-up
process. On-column cryofocusing was accomplished using
a "tee" assembly consisting of a U16-inch Swagelock tee,
10-cm 10-pm i.d. fused silica cryofocusing restrictor and
a 10-cm 0.32-mm i.d. fused silica retention gap. A six-
port dual switching valve m e o d y n e model 7040) was
employed to direct supercritical CO, either to the capillary
column or to sweep deposited fractions from the retention
gap into the capillary analytical column. A Valco model
C 14W. 1 injection valve with an internal volume of 0.1 pL
coupled with the Rheodyne valve allowed direct injection
of samples into the capillary column. A modified Hewlett-
Packard 5720A gas chromatograph equipped with a flame
ionization detector was used as the supercritical fluid
chromatograph. Separation of pesticides was accomplished
J. Microcolumn Separations, Vol. 3 , No. 1 , 1991
either on a J&W Scientific DB-1701 (14% cyanopropyl
phenylmethylsi1oxane)bonded fused silica capillary column
(20 m x 0.10-mm i.d. and 0.4-pm film thickness) or a Lee
Scientific 50% phenyl methylsiloxane (20 m x 50-pm i.d.
and 0.25-pm film thickness) fused silica capillary column.
Supercritical fluid pressures were maintained in the
capillary column by a 20-cm x 10-pm i.d. fused silica
restrictor connected to the detector end of the analytical
column via a 1/32-inch zero dead volume Swagelockunion
(11).
Procedure. The valve arrangement in the SFSPE/SFC
allowed two flow modes. In the first, needle valves V1
and V2 are open, and the six-port switching valve is in the
"extraction/direct injection" position (Figure 1). At these
valve settings, the capillary and packed columns are
isolated and allow independent use. In the extraction
process, mobile phase flows through the packed column
into the flow cell of the u v / v I s detector and finally
through the cryofocusing capillary restrictor. Supercritical
fluid decompresses in the cryofocusing trap assembly
which causes extracted solutes to be deposited in the
retention gap. Decompressed CO, gas is vented through
the Rheodyne switching valve to atmosphere.
Flow route 2 directs mobile phase through the Valco
injection valve and the switching valve into the capillary
analytical column, making direct injection of samples
possible. Valve V1 is closed and V2 is open. By switch-
ing the Rheodyne valve to the "analysis" position, the
supercritical fluid flushes deposited analytes from the
retention gap into the capillary analytical coluinn for
separation and detection.
Sample preparation. Fats, including those from
soybean oil, lard, beef fat, and bacon fat were spiked with
known amounts of pesticides. Typically, concentrations of
pesticides in these fats ranged from 0.7-1 10 ppm. Spiked
fat samples were mixed with about three parts of C,,
sorbent. Mixed samples ranging from 6-20 mg were
either placed on top of the extraction/clean-up column or
placed in an extraction cell which was connected to the
front of the extractiodclean-up column via a 12-inch x
1/8-inch i.d. stainless steel tube. Pesticide amounts ranged
from 6-100 ng.
Conditions. Typical SFSPE conditions were carbon
dioxide mobile phase (Scott specialty gases), 100°C oven
temperature, linear density gradient of 0.2 g mL-' to 0.6
g mL-' over 15 min and a final density of 0.6 g 1nL-I. The
extraction process was monitored by a Hewlett Packard
5482 UV/VIS photodiode array detector at wavelengths of
190, 250, and 300 nm. Fractionation of the extract was
done at 30 min intervals. The initial decompressed mobile
phase flow rate was 40 to 70 mL min-I.
Capillary SFC conditions were carbon dioxide mobile
phase, 100°C oven' temperature, linear density gradient of
0.2 g mL-' for 1 min to 0.6 g m t ' over 30 min and a
final density of 0.6 g mL-', and detection by FID at
400°C. The decompressed gas flow rate was 6-12 mL
m i d at the restrictor.
J. Microcolumn Separarions, Vol. 3, NO. 1, 1991
Pycriticai
6-Port
Valve
Packed
Column
Photodiode
Array Detector
FID
Figure 2. Instrumental set-up f o r packed column SFC.
RESULTS AND DISCUSSION
Three major points were considered in the design of
the SFSPE/SFC system: (1) integration of vital steps such
as extraction and clean-up, (2) use of neat C Q , and
(3) construction of a flow system which could be easily
automated for operation with minimal manual intervention.
Previous SFE/SFC systems have been designed with small
packed columns used for extract trapping. These packed
columns have not been used in a clean-up step. In this
system, packing for the small trapping column was
selected for trapping effectiveness as well as chromato-
graphic fractionation of extracts.
The second design criteria defined was use of unmodi-
fied CO, as the supercritical fluid for both the extraction
fluid and chromatographic mobile phase. This decision
was based on several facts: (1) use of modifiers would
limit the choice of detection methods and hence compro-
mise sensitivity, (2) use of unmodified CO, simplifies the
concentration step (i.e . , evaporation of modifier solvents
from the extract) and also eliminates handling of toxic
effluent wastes, and (3) the low critical temperature of
CO, allows extraction and analysis to be carried out at low
temperatures, enabling analysis of thermally labile com-
pounds.
13
A common goal of analytical chemistry is to automate
complex analysis schemes. Flow systems are used in a
number of analytical schemes to provide procedural
simplification. The design represented in Figure 1
incorporates features such as photodiode array detection of
the extractiodclean-up step which could easily be automat-
ed and used with an expert system. Future designs will
include computer-controlled pneumatic valving systems.
On-line SFE/clean-up/SFC using the apparatus sum-
marized in Figure 1 can be theoretically accomplished by
two different approaches. One method is to selectively
retain the sample matrices while eluting and depositing
analytes of interest in the cryogenic trap. The deposited
fraction could then be flushed into the capillary analytical
column for separation and detection. Eventually, sample
matrices can either be flushed from the clean-up column
stationary phase, or as an alternative, the stationary phase
can simply be discarded. The second method would
selectively retain analytes of interest while flushing sample
matrices from the stationary phase. Elution and deposition
of analytes in the cryogenic trap followed by flushing
analytes into the capillary analytical column for analysis
would complete the sample introduction sequence.
Using the first approach, investigations have been
conducted to evaluate column packings which could be
used in the clean-up column to selectively retain lipids and
allow analytes to pass through in supercritical C 0 2 . A
polarity mixture containing acetone, benzophenone,
nitrobenzene, chloroform and hexane mixed with soybean
oil in a solution with carbon disulfide was used as a test
sample. Elution properties, through various commercially
available liquid chromatography sorbents using super-
critical C02, were evaluated. The instrumental setup is
depicted in Figure 2. Results, summarized in Table I,
show that lipids can be selectively retained on all of the
column sorbents while allowing analytes to elute. In all
cases, analytes eluted before the lipids; however, on highly
polar media like silica or aminopropyl, lipids were totally
retained. The retention time differences between analytes
Table I. Packed column SFC (on diyerent cobmns) of a test
mixture containing nitrobenzene, acetone, hexane, benzo-
phenone, chloroform, and soybean oil; all dissolved in carbon
disuljde.
Retention time (min)
Sorbcnt
Test compounds Glyccridcs
Silica 10-35 > 120
Octadecyl (C,*] 5-20 - 60
Cyanopropyl (CN) 5-20 - 80
Aminopropyl (NHJ 10-40 > 120
Ethyl (C2) 10-30 - 90
14 J. Microcolumn Separations, Vol. 3, No. 1, 1991

SFC of 0 to 30 A
f minutes fraction-

Diuron
t

tSFC of 30t060~
minutes fraction

Figure 3. SFC chromatograms showing extraction of

diuron.

l---++----j
and lipids on the octadecylsilane (C,,) and cyanopropyl- 0 20 40
silane (CN) sorbents fell within reasonable working limits. Min
The C,, material was chosen as packing for further inves-
tigation. Figure 4. SFC of bendiocarb (50 ng) (A) by direct
Following evaluation of the solid phase extraction solventless injection and (B) after full cycle of extraction/-
system, the SFSPE system was coupled with a capillary clean-up and analysis.
SFC (Figure 1). Control samples of pesticides mixed with
C,, sorbent were first subjected to analysis in order to test
the efficiency of the system. Chromatograms in Figure 3
show typical extraction profiles of diuron on C,,. In all
cases, analytes of interest eluted in the first fraction (20 to SFC of 30 to 40
minutes fraction -
30 min) with an extraction efficiency which was nearly
quantitative. Figure 4 shows chromatograms of the same
amount of bendiocarb subjected to the full cycle of
extraction/clean-up/analysis(Figure 4B) and that of direct
solventless injection (Figure 4A). The average recovery
of bendiocarb was 8 4 5 4 % RSD (based on 5 runs).
Analysis of pure samples of soybean oil mixed with C,,
sorbent showed very small amounts of nonglyceride
components in the first 20-min fraction (see chromato-
grams illustrated in Figure 5). When pork lard was used, - 1 ' I ' I ' I ' I ' I I I ' I
0 20 0 30 0 30
an increased amount of non-glyceride components eluted Min
in the first 30-min fraction (Figure 6), which is believed
to be due to the sterol content in animal fats. Chromato- Figure 5. SFC chromatograms showing extruction of
graphic traces for bacon and beef fats were similar to that soybean oil (3 mg).
for lard.
Lard and soybean oil samples spiked with various
pesticides were also subjected to analysis. In all cases, pesticides in a second collected fraction. Therefore, the
lipids were completely separated from pesticides. Chro- first 30-min extraction period was sufficient to quantita-
matograms in Figures 7 and 8, which are sequential solid tively extract and separate pesticides from lipid material.
phase extraction of soybean oil spiked with alachlor, Most of the lipid material was retained on the column for
carbaryl and diuron, show no detectable amounts of greater than 60 min under the conditions employed. The
J. Microcolurnn Sepurun'ons, Vol. 3 , No. 1, 1991 15

SFC of 20 to 30 SFC of 30 to 60
minutes fraction minutes fraction
S F C of 30 to 60-
minutes fraction

S F C of 0 to 30

I minutes fraction

I I I?-
0 15 30 0 15 30 0 15 30
Min
k-t--t l--+-t+ Figure 8. SFC chromatograms showing extraction of
0 20 40 0 20
soybean oil spiked with alachlor (23 ppm), diuron (50
Min pprn), and carbaryl (42 ppm).

Figure 6. SFC chromatograna showing extraction profile

of lard (4 mg).

r - S F C of 70 to 110,

n
minutes fraction

r SFC of 0 to 30 1
minutes fraction

0 10 30 0 30 0 30
Min
Figure 7. SFC chromatograms showing extraction of
diuronfrom soybean oil.
0 20 40
Min
SFC chromatograms in Figure 9 show the analysis of
varying amounts of lard samples (4-8 mg) spiked with the Figure 9. SFSPEBFC of lard spiked with the same
same amount of Bendiocarb (5.7 ng). Chromatogram 9C amount of bendiocarb: (A) 3.8 mg lard spiked w'tli 5.7 ng
from an 8-mg lard sample shows the interference from the of bendiocarb, (B) 4.2 mg lard spiked with 5 . 7 ng of
previously observed endogenous compounds which were bendiocarb, and (C) 8 mg of lard spiked with 5.7 ng of
co-extracted with the pesticide. As can be seen from bendiocarb.
16
chromatograms 9A and 9B, interference due to non-
glyceride components is less prominent in the smaller
samples (3.8 and 4.2 mg). The average extraction effi-
ciency for bendiocarb from soybean oil is 81+6% RSD
(based on 5 runs).
Chromatogram C in Figure 9 illustrates that 0.7 ppm
of bendiocarb can be extracted and analyzed. The detec-
tion limit of the FID is 2 ng for bendiocarb. The detection
limit could be improved by employing a mass spectrometer
as a detector. Common mass spectrometer sensitivities are
100 pg full scan and at least 100 fg in the selected ion
mode.
Present limitations to the method can be summarized
as nonselectivity of the FID detector and the coelution of
nonglyceride components in soybean oil and rendered fats.
The major component of the nonglyceride fraction of
soybean oil which overlapped with the pesticide region was
identified as fatty acids. Mass spectrometry provides
added selectivity and will minimize the problem. The
major objective of the study was to develop a system for
separation and selective retention of sample matrices from
analytes, and only a minimal effort has been made to
optimize detection limits. Work is underway to interface
a mass spectrometer to the system and to investigate other
operational parameters and separation strategies to further
improve both extraction and sensitivity.
ACKNOWLEDGMENT
The authors express their thanks to the USDA for
support of this work and to Dr. John France for his
help fiil suggestions.
J. Microcolumn Separations, Vol. 3, No. 1, 1991
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