مرکزی صفحہ Chromatographia On-line coupling of packed column supercritical fluid chromatography with multiple wavelength...

On-line coupling of packed column supercritical fluid chromatography with multiple wavelength detector and radioactivity flow-through detector

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جلد:
38
زبان:
english
صفحات:
11
DOI:
10.1007/bf02269842
Date:
April, 1994
فائل:
PDF, 646 KB
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آپ کتاب کا معائنہ کر سکتے ہیں اور اپنے تجربات شیئر کرسکتے ہیں۔ دوسرے قارئین کتابوں کے بارے میں آپ کی رائے میں ہمیشہ دلچسپی رکھیں گے۔ چاہے آپ کو کتاب پسند ہے یا نہیں ، اگر آپ اپنے دیانتدار اور تفصیلی خیالات دیںگے تو لوگوں کو نئی کتابیں ملیںگی جو ان کے لئے صحیح ہیں۔
On-Line Coupling of Packed Column Supercritical Fluid
Chromatography with Multiple Wavelength Detector and
Radioactivity Flow-Through Detector
P" L e m b k e 1 / H. E n g e l h a r d t 1. / R. E c k e r 2
llnstitut for Angewandte Physikalische Chemic, Universit[it des Saarlandes, 66041 Saarbrticken, Germany
2Hewlett Packard, 76337 Waldbronn, Germany

Key Words
Supereritical fluid chromatography
Packed column SFC
Radioactivity flow-through detector
Multiple wavelength detector
Modifier gradient

Summary
The Coupling of packed column supercritical fluid
chromatography with a commercial "Radioactivity
Flow-Through Detector" for the sensitive and highly
selective detection of radioactive nuclides (e.g. 14C) is
described. The radioactivity flow-through detector
showed no baseline shift when pressure or modifier
(methanol) gradients were applied. The detector cell
Was pressure resistant even at high flow rates, over a
Period of approx. 100 hours and showed no leakage
Problems even at 40 ~ 340 bar, 4.0 ml/min and a
modifier content up to 20 %.

Introduction
In Pharmaceutical and environmental analysis continuous flow-through radioactivity detectors have been
Used for selective detection of radiolabelled comPOUnds for over 20 years [1-5]. This technique is
especially useful for the investigation of the transformation and disposition processes of newly developed
drugs, pesticides, etc. [6-8].
In principle the radioactivity flow-through detector
functions as follows: the decay of radioactive nuclides
results in beta or gamma ray being emitted. These ray
cannot be detected directly and are therefore converted in the detector cell with help of the scintillator
material into photons which are measured by a photomultiplier. Here the small photon pulses are converted
Qhromatographia Vol. 38, No. 7/8, April 1994
0009-5893/94/04 0491-11

$ 3.00/0

into relatively large electrical signals which are recorded in form of peaks.
One has to distinguish between homogeneous and
heterogeneous radioactivity flow-throug; h detectors. In
the case of the homogeneous detector the LC eluent is
mixed with a scintillation liquid before it enters the
detector cell ("liquid cell"), while in the case of the
heterogeneous detector, the cell contains a scintillator
powder ("solid cell"). Both detector types show advantages and disadvantages [12]. The homogeneous detector is to be preferred for low- and very low-energy [3emitters. The heterogeneous detector has the advantage, that no scintillation liquid is mixed with the
sample-containing eluent causing dilution and contamination. This absence of a diluent allows further
investigation of the eluent after passing the radioactivity flow-through detector. A further advantage of the
heterogeneous detector is the fact that the scintillation
material can be re-used.
Most of the published papers use the heterogeneous
radioactivity flow-through detector coupled to high
pressure liquid chromatography (HPLC). But the use
of liquid scintillation cells (homogeneous detector)
coupled to reversed phase HPLC has also been
reported [9, 10].
The on-line coupling of a radioactivity flow-through
detector with packed column supercritical fluid chromatography (pc-SFC) has so far only been mentioned
in a short note by Wilson et al. [11] who used on-line
radioactivity detection of 14C propanolol following
SFC.
In this study the separation of two polar herbicides and
their metabolites by pc-SFC and consecutive flowthrough detection will be described. The information
available on the solutes is given in Table I. The
molecular weight of the compounds did not exceed 300
dalton.
The application of pc-SFC in connection with multiwavelength detection and on-line radioactivity monitoring will be described.

Original
9 1994 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH

491

Table I. Structural parameters of solutes
Reference compounds (A)*

functional group

A1
A2

Reference compounds (B)*

=N-NO 2
-NH-C=NH
NH2*HSO~
-COOH
=N-NO 2
-OH, =N-NO 2
-NH-C=NH
NHR*HCI

A3
A4
A5
A6

functional group

B7
B8

- N H 2, - C O O H
N H M e , C O O M E , SO 2

B9
B10

- N H M e , - C O O H , SO 2
-NMe2, SO 2

* = molecular weight not above 300 dalton.

Experimental

The column was a 125 x 4 mm i.d. cartridge colum~
(Lichrospher 100 CN) with 5 gm particles (Merck,
Darmstadt (FRG)). It is important to mention, that it
was necessary to exchange the PEEK sealed cartridge
filters by PVDF filters, as the former are destroyed by
supercritical carbon dioxide (especially if methanol is
present as a modifier) within 2-4 hours.

Apparatus and Chemicals
All experiments were carried out with a supercritieal
fluid chromatograph from Hewlett Packard (Waldbronn, Germany) using packed columns. Simultaneous
UV-detecton (at two wavelengths) with a Multiple
Wavelength Detector (MWD), Hewlett Packard Series
1050) and radioactivity flow-through detection was
possible by coupling the Radioactivity Flow-Through
Detector (Model "Ramona 93" from Raytest Isotopenmessger~ite GmbH, Straubenhardt, FRG) on-line after
the Multiple Wavelength Detector (Figure 1). The
radioactive flow-through detector (RAD) was equipped
with a ll0p~l heterogeneous eel! containing glass
scintillator powder, and had a certified maximum
operating pressure of 500 bar.
Carbon dioxide (SFE and SFC Grade) was purchased
from Air Products and Chemicals Inc., Allentown
(USA). Methanol (HPLC-Grade) was obtained from
Baker Chemicals, GroB-Gerau (FRG) and isopropylamine (puriss.) from Fluka AG, Neu-Ulm (FRG).
The syringe filters with teflon PTFE membranes,
0.45 gm, were purchased from Nalgene, Rochester,
New York (USA).

1

Samples and Sample Pretreatment

.13_

I

Results

Figure 1
Schematic diagram of the SFC/MWD/radioactivity flow-through
detector system. 1 = Carbon dioxide supply, 2 = pump for CO 2
(peltier cooled), 3 = modifier pump, 4 = modifier reservoir, 5 =
packed column, 6 = oven, 7 = multiple wavelength detector
(MWD), 8 = radioactivity flow-through detector, 9 = variable
restrictor, 10 = waste jar filled with MeOH.

492

The investigated analytes in the different animal tissues
and urine (El-3) were characterized by their functional
groups and range of molecular weight, From Table I it
can be seen, that the samples are very polar, and thus
insoluble in unmodified carbon dioxide. The solubility
of most of the reference substances increased signifi"
cantly with increasing amount of methanol.
The reference substances were dissolved in pure
methanol ( 1 % solution) and were ready for chroma"
tography. Samples E1 to E3 were methanol extracts
from goat fat (El) containing 1.25 x 10 exp. 6 dpm, goat
muscle (E2) containing 3.55 x 10 exp. 6 dpm and goat
urine (E3) containing 10 • 10 exp. 6 dpm.
Each sample was evaporated to dryness and the0
dissolved in 2 ml methanol. After approx. 1 minute
ultrasonic treatment they were forced through a 0.45 mr0
PTFE membrane syringe into a sample vial. After the
gentle removal of the methanol (nitrogen stream) the
residue was redissolved in 100 gl methanol. This solu"
tion was injected into the chromatographic system.
With this sample preparation, no quantitative recovery
of the radioactive labelled analytes could be expected.
The aim of this work was not a quantitative recovery
but a successful and reliable coupling of the radioactiV"
ity flow-through detector to the pc-SFC.

Radioactivity Flow-Through Detector Cell
Stability Tests
To ensure a successful performance of the radioactivity
flow-through detector under SFC-conditions varioUS

C h r o m a t o g r a p h i a Vol. 38, No. 7/8, April 1994

Original

stability tests were carried out. Initially the pressure
stability of the detector cell was tested from 50 to 380
bar (in 20 bar steps) at 40 ~ and a flow rate of 1.0 ml/
rain. Thereafter the flow rate was increased gradually
from 0.5 ml/min to 4.0 ml/min at pressures of 200 bar
and 300 bar (T = 40 ~
The cell proved to be resistant
against the above applied conditions and showed no
leakage problems.
Supercritical carbon dioxide can be very aggressive
towards various seal materials (e.g. PEEK) especially
in the presence of methanol as modifier. Therefore the
Stability of the detector cell was investigated under
extreme conditions (20 % methanol, 340 bar and flow
rates between 0.5 and 4.0 ml/min). Even under these
Conditions no in stability or leakage problems occurred.
The radioactivity flow-through detector ran over a total
period of approximately 100 hours under typical SFC
conditions, with and without modifier and various
gradients, giving no performance problems whatsoever.
Furthermore, the base line reaction of the detector
towards pressure- and modifier (methanol) gradients
Was studied. Figure 2 shows, in the case of a methanol
modifier gradient that no effect on the base line drift
nor the base line noise can be observed. The same
behaviour could be seen for various pressure gradients.
This is an important point, as it enlarges the application
range of the radioactivity flow-through detector considerably from analytes supercritical solube carbon in
dioxide to those soluble in methanol without any loss in
Sensitivity.

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Method Development for Chromatographic
Separation
The entire optimization of the chromatographic separation was carried out with non-radiolabeled reference
compounds, and a UV detector. The radioactivity flowthrough detector was always run simultaneously with
the UV-detector during the method development to
follow its performance under various separation conditions.
Due to the polar structure of the analytes (Table I) a
moderately polar cyanopropyl column was chosen for
the chromatographic separation to ensure sufficient
selectivity without too strong retention of the analytes.
Figures 3a and 3b show the separation of the reference
samples A1 to A6 with a methanol gradient at 200 bar
and 40 ~ at two different wavelengths. The chromatogram in Figure 3a shows the applied methanol gradient
profile due to the low detection wavelength of 210 nm.
With the help of this profile it can easily be seen that
only reference substances A3 and A6 elute with a
relatively low methanol content whereas the other
solutes elute at a methanol concentration of nearly
20 %. The reason for the two detected peaks of
reference compound A6 can be explained by the
occurrence of two tautomeric forms of the guanidine
structure under the separation conditions used.
To reduce unnecessarily long analysis time, the gradient was set very steep (10 % per minute) after solute
A6 eluted. With these high methanol concentrations no

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Figure2
Effect of methanol modifier gradient on the base line behaviour of the radioactivity flow-throughdetector.
Gradient: 0 to 2 min-0 % MeOH, 12 min-20 % MeOH, 14 rain-20 % MeOH, 15 rain-0 % Meoh.
Chrornatographia Vol. 38, No. 7/8, April 1994

Original

493

~U

NV-0042.D: MWD A, Sig=210,4 Ref=450,4
A1

140

130
A5

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Figur3e
a) pc-SFC separation of reference test mixture "A" at 210 nm (MWD) on cyanopropyl column (125 • 4 mm
i.d.), 40 ~ 200 bar, 2 ml/min. Modifier: 100 % MeOH. Gradient conditions: see table 2.

mAU

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b) pc-SFC separation of reference test mixture "A" at 270 nm (MWD) on cyanopropyl column (125 • 4 mm
i.d.), 40 ~ 200 bar, 2 ml/min, Modifier: 100 % MeOH. Gradient conditions: see table 2.

494

C h r o m a t o g r a p h i a Vol. 38, No. 7/8, April 1994

Origirlgl

c o n s u m p t i o n . T h e s e p a r a t i o n o f s a m p l e A 1 - 6 with
r e v e r s e d p h a s e H P L C t a k e s up to 6 0 - 8 0 m i n u t e s a n d
r e q u i r e s a s o p h i s t i c a t e d h e p t a n e s u l f o n i c acid g r a d i e n t
s y s t e m (see T a b l e II). F o r c o m p a r i s o n t h e c h r o m a t o g r a p h i c c o n d i t i o n s o f b o t h t e c h n i q u e s a r e l i s t e d in
T a b l e II.

SUpercritical fluid c h r o m a t o g r a p h y is p o s s i b l e . N e v e r theless, w e h a v e a k i n d o f " l o w d e n s i t y H P L C " s h o w i n g
m o r e c h a r a c t e r i s t i c s o f p c - S F C t h a n L C : for i n s t a n c e
high flow r a t e s w i t h o u t t o o m u c h loss in s e p a r a t i o n
efficiency, s h o r t m e t h o d d e v e l o p m e n t a n d s h o r t a n a l y sis t i m e a n d s i g n i f i c a n t l y r e d u c e d o r g a n i c s o l v e n t

Table II. Comparison between a RP-HPLC method and the pc-SFC method for the separation of reference test mixture "A".
RP-HPLC

pc-SFC

Column

5 txm Lichrochart Superspher 100
RP18, 250 x 4 mm i.d.
Eluent flow rate
1,0 ml/min
Injection volume
200-100 Ixl
Gradient
Flask A: 15 mM heptanesulfonic acid,
1 % , acetic acid, 0.5 % in TEA in H20,
I%NaCI
Flask B: 15 mM heptanesulfonic acid,
1 % acetic acid, 0.5 % TEA in 200 ml H20,
800 ml methanol, 1 % NaCI
at 0 min 0 % B
at 5 min 0 % B
at 20min 2 0 % B
at 30min 2 0 % B
at 45 min 100 % B
at 55 min 100 % B
at 62 min 0 % B
at 80 min 0 % B
Total chromatographic
50-80 rain
----.analysis time #

5 ~tl Lichrospher 100 CN,
125 x 4 mm i.d.
2,0 ml/min
5 ktl
CO 2 + methanol (+ 0.5 % isopropylamine)*
at 0 min
7 % MeOH
at l m i n
7 % MeOH
at 2.3 min 20 % MeOH
at 10 min 20 % MeOH
at 11 min 7 % MeOH

10-12 min

* = not essential for the separation of test mixture "A".
# ~ inclusive column reequilibration time.

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Figure 4
pc-SFC separation of reference test mixture "A" at 270 nm (MWD) on cyanopropyl column (125 x 4 mm i.d.),
40 ~ 200 bar, 2 ml/min. Modifier: 99.5 % MeOH and 0.5 % isopproppylamine as additive. Gradient
conditions: see table 2.
Chrornatographia Vol. 38, No. 7/8, April 1994

Original

495

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Figure 5
Separation of reference test mixture "B" at 254 nm (MWD). 5 ~tm Lichrospher cyanopropyl column, 125 x 4 mm
i.d., 90 bar, 40 ~ flow 2 ml/min, modifier gradient: from initially 9 % modifier, at a rate of 0.6 % modifier/min,
to 20 % modifier,
a) modifier: 100 % MeOH.
b) modifier: 99.5 % MeOh + 0.5 % isopropylamine.

496

Chromatographia Vol. 38, No. 7/8, April 1994

Original

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NV-0078.D:
BIO
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Figure 6
Silmultaneous detection of reference test mixture "B" at 210 nm and 254 nm. 5 ~m Lichrospher cyanopropyl, 125
x 4 mm i.d., 90 bar, 40 ~ flow 2 ml/min, modifier: 99.5 % MeOH + 0.5 % isopropylamine, modifier gradient: from
initially 11% modifier to 13 % (0.6 %/min), from 13 % to 20 % modifier (5 % min).

Figure 3b shows the same separation detected at 270
nm. This wavelength has the advantage that the
gradient profile and minor contaminants are not
visible.
In Figure 4, the same reference sample was chromatographed under identical conditions as in Figure 3
except that 0.5 % isopropylamine was added as an
additive to the methanol modifier. The addition of the
additive caused several effects. First the analysis time
Was prolonged and then the reference peaks A6a/b
elute after A4, showing an inverse elution order
themselves (A6a before A6b). Further, the selectivity
between peaks A5, A1 and A4 is reduced after the
addition of isopropylamine although it still remains
sufficient.
The peak size of A6 is much larger compared to Figure
3b because additional analyte was added to the sample
Solution. Due to the very poor solubility of reference
A2 (ionic structure) in methanol, no A2 could be
detected in Figure 3b. After adding some more
reference~ubstance A2 to the test solution a small peak
Was obtained in Figure 4.
The separation of the test mixture "A" is superior
Without additive in respect to analysis time and
resolution. However, as sample mixture "B" required
the additive for a successful separation, it was more
COnvenient for us to run test mixture "A" with 0.5 %
additive as well.
!n Figures 5a and 5b the separation of test mixture "B"
is shown with and without additive in the modifier.
Again the additive caused an increase of the analysis
time but on the other hand, improved the selectivity
between B7 and B8 considerably by inversing the
elution order.
Chromatographia Vol. 38, No. 7/8, April 1994

The uncharacteristically bad peak shapes of solute A3
(Figure 3b), B7 and B9 (Figure 5a) can explained by
their significant polar free acid group as shown in Table
I. Even though no basic additive was used in the mobile
phase for these chromatograms, we found that it was
extremely difficult to remove the additives quantitatively from the column once they were exposed to it.
Therefore the acidic compounds of the investigated
sample are able to interact with the remaining basic
additives on the stationary phase, resulting in peak
tailing. The addition of additive to the mobile phase
results in a increased concentration of additive on the
stationary phase due to adsorption. Consequently the
retention of the acidic compounds increases as well
because of the increase in hydrogen bond acceptordonor interactions. This, in turn, tends to make the
peak shapes for acids even worse, which can be seen in
Figure 5b.
For basic compounds, on the other hand, the retention
is reduced and the peak shapes improve because the
acidic nature of the stationary phase is reduced by
adding basic additive to the mobile phase.
Through optimising the modifier gradient, it was
additionally possible to reduce the analysis time to
approx. 7 minutes, as can be seen in Figure 6, where the
advantage of simultaneous UV-detection at different
wavelengths is also shown. Solute B10 has a strong
absorbance at 210 nm but not at the usual detection
wavelength of 270 nm.

14C Radiolabeled Samples
After the method development, the radiolabeled samples (El to E3) were injected with the radioactive flowthrough detector still connected on-line after the UVOriginal

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Figure 7
Simultaneous monitored chromatograms obtained by coupling pc-SFC with MWD- and radioactivity flowthrough detection. Sample: E2 (goat muscle). Separation conditions: see Figure 3.
a) 210 nm, b) 270 nm and c) chromatogram from radioactivity flow-through detector. R = unlabeled reference
test "A"; S = Sample.

498

Chromatographia Vol. 38, No. 7/8, April 1994

Original

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Figure 8

Radioactivity flow-through chromatograms of sample E1 (goat fat) and sample E2 (goat muscle). Both
containing radiolabeled reference sample A1. Separation conditions: see Figure 3.
R = unlabeled reference test "A"; S = Sample.

multiple wavelength detector. Therefore each injection
SUpplied
hs w'~th three chromatograms (two from the
._
UV'MWD and one from the radioactive flow-through
detector) and hence interesting additional information
(See Figures 7a-c).
Due to the small injection volume of 5 lal (compared to
1 ml in LC) and the very low radioactivity of the
labelled samples, the signal of the radioactive flowthrough detector is relatively small. Nevertheless,
Pigure 7c clearly shows a peak for the 14C radiolabeled
SUbstance (A1 o) in the methanol extract of goat muscle.
In Figure 8 the radioactivity flow through chromatograms of sample E1 and E2 are compared. Both
Samples, E1 (go fat) and E2 (goat muscle), contained
the radiolabeiedt compound A1 ~ differing in their
Chromatographia Vol. 38, No. 7/8, April 1994

concentration by a factor of approx. 3. Despite the
small injection volume and therefore small absolute
amount of radionuclide in the detector cell a reliable
detection is possible. The additional introduced matrix
components had no influence on retention times in pcSFC. Figure 9a is a further example for the radioactive
flow through detection. It shows the separation of a
methanol extract from goat urine containing the
radiolabeled substance B9 ~ Obviously this compound
is metabolised, resulting in various, so far unidentified
peaks in the radioactive flow through chromatogram.
Fiigure 10 shows the radioactive flow-through chromatogram of the sample E3 obtained with HPLC coupling. The better signal to noise ratio in the HPLC
mode can be explained simply by the fact that here
Original

499

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Figure 9
pc-SFC separation of sample E3 (methanol extract from goat urine). Upper chromatogram: MWD
detection at 254 nm, lower chromatogram: radioactivity flow-through detection. Injection volume: 5 ~l,
Separation conditions: see Figure 5. R = unlabeled reference test "A"; S = Sample.

500

Chromatographia Vol. 38, No. 7/8, April 1994

Original

CPS

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Figure 10
RP-HPLC separation of sample E3 (methanol extract from goat urine). Detector: radioactivity flowthrough detector. Injection volume 1000 gl.
Gradient: Flask A: Fixanal-buffer, pH 7 + 1.5 g TBAB/liter
Flask B: Fixanal-buffer, pH 7, 60 % acetonitrile + 1.5 g TBAB/liter
at 0 m i n 9 0 % A 10%B
at30min 0 % A 1 0 0 % B
at 2 m i n 9 0 % A 10%B
at40min 0 % A 1 0 0 % B
at 20 min 60 % A 40 % B
at 42 rain 90 % A 10 % B
at 52 min 90 % A 10%B

1000 gl were injected into the system, while only 5 p.l
into the SFC system. Nevertheless, the peak pattern is
Very similar!
Comparing Figure 9 and 10, the advantages of pc-SFC
COupled with radioactivity flow-through detection are
easily seen: simple and reproducible chromatographic
COnditions and reduction of analysis time from over 30
minutes to 7 minutes.

COnclusion
The examples show the potential of packed column
SFC coupled on-line with a radioactive flow-through
detector as a very good alternative to the correspondmg LC coupling even for samples containing only trace
amounts of radioactive labelled compounds. The signal
to noise ratio can be improved simply by increasing the
injected sample volume (in our case for instance by
replacing the internal 5 ktl sample loop by an external
50 gl or 100 i.tl loop).
The radioactive flow through chromatograms show a
baseline with very little noise (0-5 cps) with modified
and Unmodified carbon dioxide. Neither pressure nor
modifier gradients influence the detector signal. Even
at Very high methanol modifier contents (20 %), where
SUpereritieal conditions are no longer present ("liquid
conditions,,), this coupling technique is superior to the
LC Coupling due to the high flow rates possible and
therefore short analysis time and short reequilibration
time of the column. Both shorten the method development time considerably and the amount of organic
SOlvents as well as their storage and deposit costs are
Chromatographia Vol. 38, No. 7/8, April 1994

reduced. The technique is applicable for all substances
which are soluble in pure supercritical- or liquid carbon
dioxide and in the corresponding methanol modified
fluids.

References
[1] J.A. Hunt, Anal. Biochem. 23,289 (1968).
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[6] M.J. Kessler, "Quantitation of Radiolabeled Molecules
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[7] T.F. Woolf, T. Change, "Recent Advances in Drug Metabolism Methodology", in: Pharmacokinetics: Regulatory, Industrial, Academic Perspectives, P. G. Welling and
F. L. S. Tsee, eds., Dakkar, New York (1988), pp 451--471.
[8] T. Grune, G. W. Siems, G. Gerber, R. Uhlig, J. Chromatogr. 533 (1-2), 193-9 (1991).
[9] T.F. Wool]', LC-GC, 7 (10), 828-30, 832, 834 (1989).
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Received: Feb 11, 1994
Accepted: Feb 25, 1994

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