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Field Desorption Mass Spectrometry.

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Field Desorption Mass Spectrometry
By H. D. &key
New analytical
methods (2)
and H.-R. Schulten[’]
Field desorption (FD) enables mass-spectrometric investigation of large organic molecules
without their vaporization. The present state of our theoretical understanding of the ionization
of these molecules in the adsorbed state on organic emitters is described. The special problems
of the technique and prospective developments in the apparatus for future analytical problems
are outlined. The present progress report concentrates on analytical studies of biochemical
model compounds and degradation products from environmental chemicals and drugs. The
method is particularly suitable for the detection and identification of submicrogram quantities
of underivatized polar substances present in complex mixtures or pre-purified extracts from
biological materials.
1. Introduction
In mid-1969 there appeared in this journal a review article
on the qualitative and quantitative analysis of organic molecules by means of a field ionization mass spectrometer (FIMS)[I]].The report mentioned field desorption (FD) of organic
molecules[21as a technique that had at that time been very
recently introduced into mass spectrometry. Since then the
F D technique has undergone rapid development. This new
branch of mass spectroscopy is applied to the analysis of
thermally labile and biologically important substances. Since
the samples need not be vaporized for analysis, the mass
of the molecular ion can in almost all cases be accurately
determined, and the elemental composition of the compound
can thus be established. In the following sections the physicochemical principles, the analytical possibilities and the problems of the F D method are summarized for the first time.
In another
a droplet of the solution is dispensed
onto the field anode from a microsyringe fitted to a micromanipulator. The advantages of this method are (i) that measured
2. Principle of FD Mass Spectrometry
The “field desorption” concept has been known for a long
time, especially through the fundamental work of Miil/er[31
and G ~ r n e r [ ~In
] . their work atoms or small molecules such
as H2, 0 2 ,or N 2 were desorbed from metallic tips into a
field ion microscope by high electric fields of the order of
1 x los to 5 x 108V/cm.
By “field desorption mass spectrometry” the mass spectroscopist today understands a special technique involving deposition of solid organic compounds on an “activated” field anode
(emitter), subsequent field desorption of the compounds in
an ionized state, and mass spectrometric analysis of these
ions. A field anode is referred to as “activated” when it is
covered with a very large number of carbonaceous microneedles.
In this technique the solid organic samples are not vaporized
into the ion sourceLs1.They are dissolved or suspended in
a suitable medium[”], usually at room temperature, and an
activated field anode (preferably a 10-pm tungsten wire with
a dense covering of microneedles) is then dipped into as concentrated as possible a solution of the sample (“emitter dipping
technique”). When the field anode is subsequently removed
some of the solution adheres to the microneedles or remains
in the spaces between them. With a mean needle length of
30pm this amounts to about IO-’pl.
[‘I
Prof. Dr. H. D. Beckey and Dr. H.-R.
Schulten
Institut fur Physikalische Chemie der Universitat
53 Bonn. Wegelerstrasse 12 (Germany)
A n q e n . Chem. infrmat. Edit.
1 Vul. 1 4 ( 1 9 7 5 ) 1 N o . 6
Fig. 1. a) Scanning electron microscope photograph of a tungsten wire 10pm
in diameter activated with benzonitrile vapor at 1200°C. Length of microneedles % 20 pm; b) Photograph of microneedles by a transmission electron
microscope. On and between the microneedles there is a thin deposit (slighter
darkening) of D-glucose which separated from a 0.01 M aqueous solution.
T h e ends of the fine needles are brought closer together by some of the
glucose threads (formed after evaporation of the water and are thereby
somewhat bent).
403
volumes of the solution can be deposited onto the anode
(e.9. for a sensitivity test), (ii) that extremely small amounts
ofsolution suffice, and (iii) that droplets of the sample solutions
can be placed accurately on the center of the field anode.
The loaded field anode is then introduced through a vacuum
lock into the ion source of a mass spectrometer. The solvent
evaporates and the substance to be analyzed remains behind
on or between the microneedles.
Fig. 1 a is a photograph of a clean activated tungsten wire,
taken by means of a scanning electron microscope. Fig. 1 b is a
transmission electronmicroscopic photograph of microneedles
on and between which D-glucose has been deposited from 0.01 M
solution. Although only about
g of D-glucose was deposited, an F D mass spectrum suitable for interpretation could
be obtained. The detection limit of adenosine solution for
analysis by the emitter dipping technique is about
M;
this means that about to-" g is the smallest amount of an
organic substance that can be detected by FD-MS when using
a modified Varian MAT-CH4 mass spectrometer. This limit
depends, on the one hand, on the nature of the substance,
the solvent and its pH, and the morphological structure of
the microneedles, and on the other, on the transmission of
the mass spectrometer (see Section 8.1) and the sensitivity
of the ion-detection system. If substances present in concentrations smaller than I O - ' M are to be analyzed then larger
volumes of solution must be transferred to the field anode;
for example, the field anode can be gently heated so as to
evaporate the solvent. If the heating current just compensates
the heat of evaporation of the solvent, no thermal decomposition of the sample will occur[']. Since volumes of the order
of lo3 greater than by the "dipping technique" can be loaded
in this way, solutions down to about
M can be investigated.
The sensitivity of the F D method (expressed in coulombs
of collector current per pg of field-desorbed substance) is
only slightly lower than that of the electron-impact ionization
method (EI method) for systems with direct vaporization of
solid organic substances. The relatively high sensitivity of
the F D method is due to the ionization efficiency and, further,
to the efficiency of the substance supply processes. A large
fraction of the molecules adsorbed on the emitter is desorbed
in the form of ions, and one can expect that, as a result
of the field distribution in front of the microneedles, a large
portion of these ions will be emitted in the direction of the
counter electrode. As yet no measurements have been made
of the angular distribution of field-desorbed ions on acticatrd
emitters; only the angular distribution of the field ion current
at individual metal tips has been measured[8.91.
For recording an F D mass spectrum the field anode is
first adjusted mechanically by means of a micromanipulator
(mass spectrometers of Type Varian MAT CH4/CH5, AEI
MS9, and CEC21-IIOB modified by the authors) or alternatively brought through a vacuum lock system into a mechanically fixed and reproducible position in the ion source (commercially available mass spectrometers of Type Varian MAT
CH5, 711, 731). The ion beam is then focused electrically,
which involves loading the field anode at room temperature
(so that no solid substance is field-desorbed), and admission
of acetone vapor into the ion source which is used in the
FI mode. The admission of acetone is stopped after optimization of the field ion current. The field anode is normally
brought to a positive potential of 3-10 kV with respect to
404
ground and the counter electrode, at a few mm distance,
is brought to a negative potential of a few kV, so that the
total potential difference between the field anode and the
counter electrode amounts to 10-12kV. The current due
to ionization of the solvent (e.g. acetone, ether, benzene, water)
soon ceases on gentle heating of the field anode. A small
residual current of solvent ions does not interfere with the
subsequent measurements. Characteristic F D mass spectra
of relatively volatile organic substances can be obtained with
the field anode at room temperature or slightly above. However, in the case of most polar organic solids the production
of a sufficiently intense FD mass spectrum requires stronger
heating of the field anode (heating currents of the order of
10-40mA for activated 10pm tungsten wires).
It should now be explained qualitatively why organic
samples suffer less thermal stress in the F D technique than
in field ionization (FI), electron-impact ionization (El), or
chemical ionization (CI). In the last three techniques the solid
samples must be vaporized; this requires that energy equivalent
to the total sublimation energy of the samples must be supplied.
In the F D method, on the other hand, ionization of the
adsorbed molecules and desorption of these ions from the
field anode demands only the field desorption energy Qder.
which is small at high field strengths. This is shown schernatically in Fig. 2 for an adsorbed atom (for molecules the situation
is considerably more complicated). Fig. 2a shows the potential
curves for the interaction of an adsorbed atom or atomic
ion, respectively, with a pure metal surface MI4]. It is assumed
here that the ionization energy I of the atom A is appreciably
greater than the work function of the metal. For the conversion
of the adsorbed atom into the ion A ' an energy EdL,=
QdL.,+I -@ (normally of the order of several eV) must be supplied. When a strong electric field is applied ( 21 V/A) the
distorted potential curves shown in Fig. 2 b are obtained:
an appreciably lower energy Qhe.;is required for the formation
of the ion A'. At extremely high field strengths the atom
can thus bedesorbed into the ionized state without the application of thermal energy. The theory of field desorption of
moms from pure metal surfaces has been treated, inter aliu,
by Miillrr and Tsong["].
0
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a)
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Fig. 2. a ) Potential curves for the interaction of a n atom ( A ) o r a n atom-ion
( A ' ) with a solid metal surface ( M ) in the absence of a n external electric
field ( @ = w o r k function of the metallic emitter): b) Potential curves in the
presence of a strong electric field. Smaller polarization terms have been
neglected. e F x = potential of external field. F Abscissa: x=dtstance of atoms
For other designations see text.
from the metal surface in
A
There isas yet no quantitative theory for the field desorption
of large organic molecules from microneedles of carbonaceous
organic polymers with structured and graphitized regions" ' I .
Anyeit.
Chrm. inl~l'iiur Edil
Vol 1 4 f l Y 7 5 i
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No 6
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Fig. 3. a ) Potential curves for the interaction of a molecule ( B ) or a molecular
ion (B') with the surface (0)of a solid organic material in the absencc
of a n external electric field (@'=work function of the organic emitter): b)
Potential curves in the presence of a strong electric field. Field desorption
energy Q , must be supplied for the desorption of a n ion adsorbed at x,.
and energy Q. for the desorption of a neutral molecule adsorbed at x.
(x, is the point of intersection of the curves for the neutral and ionized
states of the molecule). Repulsion of the potential curves on the basis of
the noncrossing rule, in contrast to Fig. 2 b, has not been taken into account
in
Fig. 3b.
The observed complicated phenomena can be explained only
qualitatively on the basis of Figs. 3a and 3b. The following
effects, inter aka, should be pointed out:
1. Only some of the organic molecules are chemisorbed
on the surface of the field anode. The other molecules which
are bound to the surface by van der Waals and dipole forces
are more easily field desorbed. The potential trough for
these neutral molecules (Fig. 3a) is thus considerably shallower
than that shown in Fig. 2a.
2. in a strong electric field the potential curve for the ionic
adsorption state is shifted by an amount AV to lower potentials.
Most of AVcan probably be attributed to the potential drop
in the microneedles, a further part to the penetration of the
electric field into the microneedles.
3. Because of the low-lying localized energy levels, the electron work function of organic emitters can be greater by
a few eV than that of metallic emitters['21.
Owing to these three effects the potential curves for the
neutral and ionized states of the molecule may intersect one
or more times (Fig. 3 b).
3. Field Anodes for FD Mass Spectrometry
The best results in F D mass spectrometry have been
obtained with microneedles 20-4Opm in length (cf. [13]).
On the one hand microneedles as long as possible favor the
deposition of the sample to be analyzed, since a large surface
area is then available for adsorption; on the other hand,
increasing needle length is accompanied by a decrease in
the field strength in the emission centers and thus in the
intensity of the ion currents produced. The needle length
mentioned above has proven to be the best compromise.
Several activation methods for the preparation of microneedles have been developed by the research group at the
University of Bonn['41.
Needles produced from benzonitrile at room temperature
are often destroyed by chemically aggressive substances, particularly at high temperaturesfts1.Field anodes activated at
higher temperatures are chemically more stable" 61. Schulten
er al. developed a high-temperature (HT) activation technique
that produces microneedles which are extremely resistant both
Anyt~w. Chem. fiuernuf. Edit I Vol. 1 4 ( 1 9 7 5 ) i
No. 6
chemically and
['I. Such microneedles make
it possible, inter aiia, to calibrate the mass scale by means
of perhalogenated compounds such as perfluorokerosene or
perfluorotributylamine, which destroy needles produced at
room temperature.
It should be emphasized that FD mass spectrometry of
organic substances stands or falls on the quality of the ion
emitter.
4. Optimal Field Anode Temperature for Field Desorp
tion
Finding the optimal field anode temperature T* (often called
also "best anode temperature" (BAT) in the literature) is very
important in F D mass spectrometry. Because of the temperature gradients between the anode wire and the needle tips,
T* is not identical with the temperature of the carrier wire.
In the case of direct electrical heating of the anode T* is
usually characterized by quoting the heating current.
With certain substances greater absolute and relative molecular ion currents can be achieved by heating the microneedles
by thermal radiation["! This indirect heating of the field
anode also has the advantage that after calibration with NTC
resistances the mean temperature of the emitter surface can
bedetermined to within ca. f2"C[201.At the optimal temperature T* of the field anode the intensity of the molecular
ion current is maximal and fragmentations minimal. T o obtain
thermally-/field-induced fragmentation the work is carried out
at temperatures above T*[23!
Determination of T* will first be discussed for the case
in which the F D spectrum is determined with electrical recording and a slow mass scan without use of the multiscaling
technique (see Section 5). When the molecular weight of a
sample is to be determined, the optimal anode temperature
is the result of a compromise: if the field anode is heated
too strongly, the sample vaporizes far too fast for a complete
mass spectrum to be recorded; if the heating is too weak
the field desorption of the sample is insufficient for the production of an ion current that would give rise to signals that can be
clearly distinguished from the background noise of the ion
detector.
T* (expressed in mA of heating current) depends on the
wire diameter, the effective field strength, the nature of the
substance, the solvent and its pH, the layer thickness of the
substance deposited on the field anode, and particularly on
the length and structure of the microneedles. If the substance
is insoluble it can be transferred onto the emitter from a
suspension in readily volatilizable liquid[21!
T* is determined by first setting the mass range of the
molecular ion group (which is almost always dominant in
field desorption) on the mass spectrometer. Several mass scans
are then made through this region while slowly raising the
temperature of the field anode. An intense and relatively well
reproducible F D mass spectrum is often obtained only within
a narrow temperature range. For some substances T* is equal
to room temperature (e.g. for naphthalene, phenanthrene and
Endrin, i. e. substances having relatively high vapor pressures);
I'[
Reproducible HT activation of field anode wires is described
in
Ref.
[ 1 7 ] . A n oxidation-reduction procedure [I81 for the tungsten wire before
activation becomes unnecessary if the parameters cited
all strictly observed.
in
Ref. [I71 are
405
for others T* may be several hundred degrees centigrade,
i e . the field anode must then be heated with an emitter
heating current of 10-40mA.
5. Detection of Ions on Photographic Plates or by Rapid
Electrical Recording
The disadvantages of the F D technique (small absolute
ion current intensities, unknown optimal temperature T* with
samples of unknown composition; see also Section 7) can
be overcome by integrating ion detection with photographic
plates or by rapid electrical recording using the multiscaling
technique.
When photographic plates are used in a double-focusing
mass spectrometer, the optimal emitter temperature can be
determined by heating the field anode until the ion current
at the counter electrode amounts to about 5 x 10-8A (for
8 k V acceleration potential and d=2mm)[221.Because of the
simultaneous recording ofall types of ions on the photographic
plate, no significance attaches to the variations in and the
general decrease of the ion current due to consumption of
the substance. This fact is even more important for the F D
than for the electron-impact method. Furthermore, with photographic plates one can record an FD mass spectrum at
temperatures somewhat above T*; not only the molecular
ion group but also numerous fragment ions then appear'231
and enable conclusions to be drawn about the functional
groups or branching sites in an unknown organic molecule.
An important application of F D mass spectrometry with photographic recording of the ions lies in a range well above
T* where pyrolytic products are detected r. y. in the pyrolysis
of polymers (see Section 8.2).
Alternatively, rapid electrical recording of the ions may
be undertaken instead of detection with photographic plates.
In the multiscaling technique the scan through the mass spectrum is so fast that the intensities of the various ions do
not change appreciably during a single scanning period. Since
only a few ions per second reach the ion detector, use must
be made of counting techniques e.g. with a multichannel
analyzer. It is more advantageous to use a process computer
which controls the mass scan through the magnetic field,
stores the ion types, and programs the time/temperature profile
of the field anode. In our laboratories we use a PDP 8/E
process computer (Digital Equipment)['].
possible intensity, since in many cases determination of the
molecular weight and the empirical formula of an unknown
compound are the main aims of MS investigations.
4. The procedures for qualitative and quantitative analysis
must be reliable even if the samples are thermally unstable.
6.1. Analysis of Pesticides
In recent years mass spectrometry has proved its value
in the qualitative and quantitativedetection of toxic substances
occurring in the environment. It has proved particularly valuable when used in combination with gas chromatography
and with rapid electronic data recording and data processing
by computer (libraries of spectra, teaching machines).
6.1.1. Chlorinated Polycyclic Hydrocarbons
Fig. 4a shows the electron-impact ionization (EI)mass spectrum of Endrin (a widely used insecticide, acaricide, and rodenticide)lZ5! The molecular ion at m/r 378 is recorded with
about 1 relative intensity. In the FI mass spectra determined
by Damico et
for a series of chlorinated polycyclic
hydrocarbons the molecular ions of these compounds made
up a major portion of the ion current produced. Endrin is
sufficiently volatile for a field ionization mass spectrum to
be obtained at 30°C; in addition to the intense molecular
ion group (base peak) a signal at m/e 342 was observed with
about 10 "/, relative intensity. The corresponding mass lines
for the metastable ions provide evidence of loss of HCI from
the molecule. Fig. 4 b shows a field desorption mass spectrum
recorded at room temperature with electrical detection and
at low resolution (modified Varian MAT-CH4 mass spectrometer)'26];it consists exclusively of the molecular ion group,
no fragments being detectable. Under the quoted experimental
conditions FD spectra containing only the molecular ions
are also obtained from other pesticides of the dimethanonaphthalenr type, such as Dieldrin, Aldrin, and I~odrin[~'!
'x
ill
6. Application of Field Desorption Mass Spectrometry
Mass spectrometry has found ever increasing use in the
field of biochemistry over the last four years. Especially strict
requirements must be met for analytical investigations in this
field and in environmental research and medicine:
1. The detection limit should lie within or below the nanogram region.
2. The selectivity of the method should permit unambiguous
detection of substances in only partly purified crude extracts
of biological material in the presence of unavoidable impurities.
3. The mass spectra should not only show the characteristic
fragment ions but also the molecular ions at the greatest
[*] The programs for carrying out these operations were compiled by
C'. Winklrr. Bonn (Germany).
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Fig. 4. a ) El mass spectrum of Endrin; sample temperature 55°C [25];
b) FD mass spectrum of Endrin; OmA emitter heating current :30"C.
Both spectra were recorded electrically.
The only F D mass spectra so far published for insecticides
of thedimethanoindene type are those of Chlorden, Heptachlor,
Endosulfan. Kclevan. and Keponc'". '"I. A comparison of
the EI and FD spectra of Chlorden shows that, under identical
experimental conditions. there is again the typical picture
ofthe fragment-free spectrum with F D and of intense fragmentation with El. However. unlike tndrin; Chlorden is not
dcsorbed optimally at room temperature, T* being at slightly
elevated emitter temperatures (8 mA emitter heating current
~50°C).
The lower Iolatility of Chlorden is also shown in
the El spcctrum, as above m/e 66 there are only two peaks.
at mje 101 and m;c 237. with more than 3"/,relative
int~nsity''~!
6.1.2. C h l o r i n a t d Diphcnylmcthanes
The widespread, and extensively used insecticides of the
chlorodiphenylmethanc type may be mcntioned a s examples
that illustrate the behavior o f leading pesticides under the
conditions of field dcsorption. O n using photographic detection at a resolution of (.a. l5ooO (lo');, valley definition) the
parent substance p.p'-DDT gives the FD spectrum shown
in Fig. 5"". With the aim of producing thermally-:fieldinduced fragmentation this F D spectrum was obtained with
an elevated cmitter tcmperaturc(5 mA emitter heating current).
If the F D mass spectrum of p.p'-DDT is produced at room
temperature then almost only the molecular ion group is
rccorded with either electrical o r photographic detection. In
the El spectrum this group appears with a relative intensity
o f less than SIX,, as do the pcaks at m/c 317 and m/e 282
( M -(I: M - 2CI)' [ 2 " 1 . The loss of one or two chlorine atoms
from the D D T molecule is shown more clearly on FD. but
the dircct bond fission between C ' and C 2 is still more marked
at m.'e 235 (base pcak in thc El
Since a relatively
intense pcak is also obscrvcd at M:'P I17 for (CC13)'. a typical
fcature of many F D spcctra becomes recognizable. namely
the frequent fission of the investigated molecule into two
complementary. charged particles. This direct bond cleavage
apprcciably simplifies interpretation of the spectrum.
l!O
.50
200
250
m/e
300
350
---.-_)
Fig. 5 FI) mass spectrum of p.p'-DDT [ Z X ] .
Doubly charged ions such a s M 2 - at m:e 176 and ( M CCI.3)z'at m;e 117.5 are of special interest with regard to
the theoretical aspcctsof their formation in a high field. Ro//gen
and Hec~kc~j~~3n-"1
havc shown that in F1 mass spectrometry
intermediate surface bonding of singly charged ions is always
a charactcristic of the formation of doubly charged ions. The
corresponding FD processcs are complicated by the solid
adsorbate layer. For the doubly charged ions that are formed
by bond cleavage and:or elimination of neutral particles from
the initial molecule one often finds a n intense peak at a
mass that is higher by one mass unit. Accurate mass mcasurements on sultam derivative^[^'^ havc shown that protonation
in the adsorbed layer must bc responsible for the appearance
of the second charge. Very recent FD measurements by Games
et a/.'33.341on alkali metal salts of bile acids (e.9. sodium
glycocholatc) showed doubly charged ions of composition
( M + 2 Na)2 I . In field dcsorption of organic molecules it is
thus generally better to speak of the transfer of a cation . i. e.
"cationization"-. instead of protonation[' 351. This process
is particularly important from a n analytical point of view.
since the addition of e.q. an alkali metal cation t o a large
organic molecule containing nucleophilic groups produces
stable even-electron ions. whereby the possibility of ionization
and of detection of the intact molecule is increased'")l.
6.1.3. Organic Phosphorus Compounds
The potentialities of the F D method become particularly
apparent when we compare the El. (-1. FI. and F D spectra
of some pesticides such as Delnav. Phospharnidon. and Temik.
Fales et a/.'36'were able to show that in fact the decisive
advantage of field desorption lies in the comparatively mild
thermal treatment. T h e €1, C1, FI, and F D spectra of Delnav
are shown alongside one another in Fig. 6[361.I t will be
seen at a glance that with all the ionization methods a signal
is recorded in thc high mass region at m!e 270 (271 for CI)
[for the process (M-(EtO)PS2H)']: the molecular ion is
shown on/! in the FI) mass spectrum-and as the base peak
of the spectrum.
6.2. Analysis of Pesticide Decomposition Products
Why is F D mass spectrometry so particularly suitable for
the investigation of metabolites?
I . Experience has shown that the starting compounds,
whether pesticides o r drugs, are metabolized into more polar
substances (e.g. in higher organisms so as to increase their
solubility in water); their volatility ( I . e. the readiness with
which the intact molccule can be vaporized) is thereby decreased. As metabolism progresses the original substance thus
becomes increasingly unsuitable for direct investigation by
a simple combination of pas chromatography and mass spectrometry. A coupling or combination of liquid chromatography and F D mass spectrometry. however, has proven positively
ideal for such investigations.
Degradation products of pesticides containing carboxyl
groups.
e. g.
hexachloro-5-norbornene-2.3-dicarboxylic
acid'"' (from Isodrin). kelevanic acid1391(from Kelevan). photoaldrindicarboxylic acidlzxl(from Aldrin) and 2.2-bis(p-chloropheny1)acetic acid12"' (from p,p'-DDT) have been subjccted
to a comparative study by El and F D mass spectromctry.
Similarly, mctabolites containing hydroxyl gl-oups have been
analyzed. e. 9. 2.2-bi~(p-chIorophenyl)ethanol[~~~,
1-hydrouychlorden[-"l, aldrin-4.S-ci~~rrans-diol'~~~.
and the isodrin-4.5cis-diol shown in Fig. 7.Thc F D spectrum of the last-named
compound illustrates particularly clearly the small effect of
the hcat treatnicnt in field desorption: thus, in spitc of the
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relatively high intensity of the ion current that is necessary
for the high resolution of 28500 (10% valley definition), the
loss of water that might be expected in the thermal process
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408
is not observed. In addition, it is important to point out
that field desorption with photographic recording provides
a resolving power comparable in order of magnitude with
that obtainable by other methods: for EI with the commercial
ion source the CEC21-110B mass spectrometer used affords
a resolution of ca. 32000 and for FI with a special ion source
one of ca. 30000[71.
2. Since the degradation products of living organisms are
always produced in very small quantities, the sensitivity of
the detection methods assumes decisive importance. The limit
of detection by F D mass spectrometry lies in the region of
to 10 “g. However, it must be expressly emphasized
that these data refer to the amounts of substance adsorbed
on the surface of the FD emitter. It is true that the quantities
referred to suffice also for other mass-spectrometric methods,
but with field desorption there is no need for derivatization.
(For the sensitivity of the analysis see Section 2.)
3. The analytical samples available in metabolic studies
are almost without exception mixtures. For the analysis of
mixtures field ionization is recognized as being particularly
Angel\. Chrm. intrmat. Edit.
1 Vol. / 4 ( 1 9 7 5 ) 1 No. 6
well suited", 3 7 . 381 Field desorption, however, can also be
used for the qualitative analysis of mixtures.
Pesticides used in practice often consist of several active
constituents. For example, the acaricide Tetradifon is used
together with the insecticide Carbaryl[*]. Fig. 8a shows the
F D spectrum of Tetradifon. This relatively volatile substance
affords only the molecular ion group and a peak at m/e
177 for M 2 + The F D spectrum of Carbaryl (Fig. 8b) als6
shows the molecular ion as the base peak, but together with
several significant fragments at m/e 143, 101, 58, and 4SL4'].
For comparison i t may be mentioned that the M t ion in
the El spectrum appears with a relative intensity of less than
5 %, and in the FI spectrum (source temperature 40°C) with
50% relative intensity[24! If one records the F D spectrum
of the above mentioned mixture of active substances on a
photographic plate with an emitter heating current of between
0 and 30mA and 12min exposure time, then the spectrum
shown in Fig. 8c, with a resolution of IOOOO is obtained.
It can be recognized at once that this spectrum is composed
approximately additively of the F D spectra of the individual
components. Admittedly one must take into account reactions
of the individual components with one another and with
1772t
1
the emitter surface,as has been shown in more recent investigations on mixtures of
and inorganic
After
calibration of the F D spectra with standard concentrations
of Carbaryl and Tetradifon the composition of the mixture
can be estimated (here 4 :
Qualitative agreement of the
F D spectra with the composition of mixtures has been confirmed by Rinehart er a/,[421for antibiotics of complex conifor steroid conjugates, Games
position, by Adlercreurz et
et
for bile acids, and recently Schulten and Schurath
for the complex compositions of aerosols[72!
4. Pesticides and drugs are often excreted as conjugates
with amino acids, peptides, sugars and sugar derivatives,
organic and inorganic acids, erc. By using model compounds
it has been shown that F D mass spectrometry is particularly
suitable for the analysis of such conjugates. The most extensive
F D investigations carried out to date have been concerned
with this aspect (see Sections 6.3 to 6.6)[441.
6.3. Analysis of Sugars and Sugar Derivatives
6.3.1. Oligosaccharides
The first substance measured by F D mass spectrometry
was the monosaccharide D-ghcose[21;Krone and Beckey have
reported the F D spectrum of the disaccharide c e l l ~ b i o s e [ ~ ~ !
The F D spectrum of a triaminotrisaccharide is shown in Fig.
9"l; although this compound contains seven free hydroxy
and three free amino groups, the cationized (protonated) molecule provides the base peak; the peak with the second-highest
relative intensity, at m/e 324, can again be ascribed to direct
bond fission. Structurally related oligosaccharides with antibiotic activity. such as gentamycin and tobramycin, also provide high molecular ion intensities and undergo bond fission
at the glycosidic oxygen. Rinehart et a/.[421have obtained
the F D spectra ofa series of antibiotics. Neomycin, streptolydigin, and novobiocin, which afforded no molecular ions in
the El or CI spectrum and showed the molecular weight
as the base peak in the F D spectrum at T*. Furthermore,
field desorption provides qualitative indications on the composition of complex antibiotics such as streptovaricin, filipin,
and dermostatin.
200
M'
C)
ll?
M'
60101
Fig. 9. FD mass spectrum of a triaminotrisaccharide
Fig. 8. a ) F D mass spectrum of Tetradifon [41]; b) F D mass spectrum
of Carbaryl [41]: c) F D mass spectrum of a mixture of active constituents
containing 14% of Tetradifon and 50% of Carbaryl: solvent acetone: photographic recording, exposure time 10 min. optimal emitter heating current
14mA [41].
[*I We are indebted t o Dr. Schinkel of the Biologische Bundesanstalt,
Braunschweig (Germany) for supplying this mixture of active substances.
Anyrw. Chem. intrmat. Edit. / Vol. 1 4 ( 1 9 7 5 )
!
No. 6
Fig. 10a reproduces the 70eV EI and Fig. 10b the 18eV
EI spectrum of underivatized neomycin B. The spectra terminate in the higher-mass region at m/e 446/447; astonishingly,
no ion of this mass appears in the F D spectrum (Fig. 10c),
but only relatively weak fragments at m/e 206, 307, and 455,
while the ( M + H ) + ion (m/e 615) makes up 75% of the total
ion current. To obtain a mass spectrum of this compound
409
0
1003
b)
90 j
8Oj
70i
145
I
i
601 I
50
100
150
200
250
300
loo]
goa
350
450
400
500
600
550
650
m/e --+
jBS,MH,
c)
801
I
I
I
I
I
i
,
50
100
1*83.10(
,
i
458
307
of.,.,,,,,.,.3
8,
206
,
,
,
,
/
.
I
150
.
,
,.,,,
200
1
)
#
,
250
,
300
350
500
450
400
550
,
,-
600
650
m/e-
Fig. 10. a ) El mass spectrum of neomycin B. electron energy 70 eV : b) El mass spectrum of neomycln B. electron energy 18 eV :c ) FD mass spectrum of neomycin B [42]
with EI and using the direct insertion system it was necessary
to prepare the hexa-N-acetylheptakis(0-trimethylsilyl)derivat i ~ e ' ' ~ ' this
; is difficult, and furthermore the protecting groups
make the derivatized substance more than twice as heavy
and thus bring it close to the limit for analysis with conventional mass spectrometers.
ties in extracts of the natural product (e.g. after column chromatography), and with higher emitter-heating currents also
100,
I
6.3.2. Glycosides
For all spectroscopic methods there is an approximately
reciprocal relationship between the signal intensity and the
resolution. Because of the relatively small ion currents in
field desorption, high-resolution mass measurements make
severe demands on the transmission of the mass spectrometer
and on the quality of the emitter (see Section 3). The advantages
of high resolution are as follows: accurate determination of
the molecular weight and thus of the empirical formula, as
well as important indications for the interpretation of fragments. High-resolution data for some aryl glycosides and
for glycosides of the coumarin and flavone series have recently
been published[471.Fig. 11 shows the high-resolution FD spectrum of hesperidin; this also makes it clear how the molecular
ion is stabilized by cationization; M t is certainly recognizable
at m/e 610, but, unusually for FD, with less than 20 relative
intensity. In contrast, addition of N a + appears to provide
the base peak. The sodium ions arise from unavoidable impuri410
, I
3
50
I00
I50
,
,
,
,
,
,
200
,
,
250
m,e
,
,
-
,
,
,-,
3w
IMf"N.1
633
\
m
rn(.
I
350
-
Fig. 1 I . F D mass spectrum of hesperidin [47].
from impurities in the emitter. The usefulness of these
( M t N a ) ' and ( M t K ) ' peaks for differentiation of M ?
from (M + H ) + ions should be noted[48'. Since high resolution
affords the elemental composition of the fragments, m/e 302.079
can be interpreted as (A+H)+ and m/e 147.066 as due to
direct bond fission at the glycosidic oxygen of the terminal
sugar unit. The aglycone also appears as a doubly charged
ion at m/e 151.040. The EI spectrum of free hesperidin affords
a peak for the aglycone (m/e 302) with 76% relative intensity,
and the base peak lies at m/e 137; the molecular ion at m/e
610 is not detectable.
Clearly different F D spectra were obtained for CL- and plinked glycosides containing an aromatic aglycone, used as
test substances for enzyme determination (substrate for glucosidases). Comparison with EI and FI spectra showed that
the FD spectra are the simplest and provide the greatest
differences between the s t e r e o i ~ o r n e r s ~ ~ ~ ~ .
6.3.3. Glucuronides
Steroid glucuronides are extremely important for hormone
excretion. Fig. 12 shows a high-resorution spectrum of testosterone glucuronide (sodium salt). Investigations by Adlercreuti
et u1.[431have gone beyond studies with model substances
and have placed field desorption in the service of clinical
COiNa
6.3.4. Nucleosides and Nucleotides
These compounds come into consideration, firstly since
they form conjugates or take part in metabolic processes,
and are therefore of interest for use underivatized in F D
analysis, and secondly because a large number of modified,
biologically active nucleosides have been
whose
FD spectra could simplify structural determination. The most
important nucleosides that occur as constituents of RNA and
DNA have been measured as model compounds by FIL5' I
and FD[221
spectrometry. Except for guanosine, the molecular
ion intensities in the FI spectra suffice to identify the underivatized compounds. The temperature given15 for the direct
introduction system (250 "C) clearly leads to thermal decomposition of the guanosine molecule before it is brought into
the gas phase [m.p. 240°C (dec.)]. The FD spectrum[221of
guanosine was determined above T* at 18-20mA with the
intention of obtaining the complementary fragments-important for the determination of structure-for the sugar portion
(S) and the base portion (B); the molecular ion then appeared
with ca. 20 "/, relative intensity. The marked influence of the
solvent in F D was also discussed in the same paper.
Fig. 13 shows the high-resolution F D spectrum of adenosine.
The peaks for (B+H)+ at m/e 135.055 and for (S) at m/e
233.050 are characteristic, along with the base peaks for M
H
267097
\
1*611)1
mle
Fig. 12. F D mass spectrum of testosterone glucuronide (sodium salt)
1471.
chemistry and medicine. Thus, estriol 16-glucuronide isolated
from pregnancy urine could be detected with high molecular
ion intensity by FD mass spectrometry. This detection was
effected without derivatization at low resolution (m/Am 500600), and was supported by F D measurements on synthetic
reference compounds.
31
on
OH
I
CH'O
j
l
10
50
I00
-
200
I50
mle
250
Fig. 13. F D mass spectrum of adenosine [ 2 2 ] .
100
1
IM+lf
8 1
135 I 8 1
n
z
Y
I N+ H I
OH
OH
L
m/e
-
360
Fig. 14. F D mass spectrum of adenosine monophosphate r221.
Atiyrw.
Chern. inrrrnar. Edir. ;Vol. 14 ( 1 Y 7 5 ) / N o . 6
41 1
and (M + H)+ (emitter heating current 20 mA). The first free
nucleotide detected by F D mass spectrometry was adenosine
5'-monophosphate as the intact protonated molecule ion
( M + H ) + (73% rel. intens.)["] (Fig. 14). Bond fission between
the phosphoric acid moiety and the nucleoside (N) adenosine
was recorded with 2Yx) relative intensity at m/e 267.097. The
remaining fragments yielded intense mass lines for (S)+ and
( B + H ) + , as in the case of the nucleoside. Relatively weak
signals at m/e 60.021 (CHOH): and 61.029 [(CHOH)z+ H I +
provided evidence that the compound was a riboside. With
emitter heating currents well above T*, e.g. up to 50mA,
protonated orthophosphoric acid was detected at m/e 98.985.
Other free nucleotides such as the 5'-monophosphates of thymidine, deoxycytidine, and deoxyguanosine gave similar F D
spectra.
at mje 158 is characterized by loss of ammonia from the
protonated molecule [(M H t N H 3 ] +.In addition, the bond
between the guanidino residue and C 5 is broken, leading
to m/e 116 and m/e 117. With a-amino acids (e.g. Glu, Val,
Pro, Ser, and Cys) a peak is generally obtained at (M-45),
which can be interpreted as [(M+H)-COOH2]+.
+
6.5. Analysis of Peptides
It was first shown for the simplest dipeptide, glycylglythat application of field desorption to underivatized
peptides affords complete mass spectra in which, with an
emitter heating current of 18-24mA, the molecular ions
provide the base peak. In a subsequent F D investigation
Winkler and B e ~ k e y I were
~ ~ l able to record spectra from partially protected and free oligopeptides containing three to
nine amino-acid residues (including for example arginine and
histidine), not only with high molecular ion intensities but
also with some information about the amino-acid sequence.
The F D spectrum of Ac-Gly-Arg-Arg-Gly-OMe is reproduced
in Fig. 16 as example. Although the N-terminal group of
6.3.5. Vitamins
The 70eV and 12eV EI and the high-resolution F D spectrum of vitamin C (ascorbic acid) were measured in connection
with the problems of determining mutagenic compounds[281.
In this investigation the intensity of the M f ion in the El
spectrum had a relative intensity of 10%. The base peak
in the F D spectrum was formed by the molecular ion group
M'/(M+H)+ when the spectrum was determined at T*.
The characterization of several B vitamins has recently been
reported[52!
g
50-
!i
,
I):
6.3.6. Sodium Salts of Deoxyfluoro-D-glucose 6-Phosphates
217
1 1I
22L
257
'
j
136
1
At theend of this listing of compounds in increasing polarity
come the alkali salts of the sugar phosphates. The high-resolution F D spectra of these substances[s31contain ( M + H ) + as
the base peak but disclose also other products that are formed
by replacement of H + by Na+, elimination of water, and
pyrolytic processes. Since these salts are desorbed within narrow time and heating current intervals, they can only be
recorded photographically. Tarry residues deposited on the
F D emitter (0-50 mA heating current, 10min exposure time)
strongly reduce the emission properties of the emitter.
6.4. Analysis of Amino Acids
A series of biologically important a-amino acids has been
studied by F D mass spectroscopy by Winkler and B e c k e ~ J ~ ~ l .
The F D spectra usually show the molecular ion or the quasimolecular ion with relatively high intensity. This is also the
case for arginine and cystine, whereas these ions are not
detectable by EI, CI, or FI methods. The F D spectrum of
arginine is shown in Fig. 15: the ( M + H ) + ion appears with
30% relative intensity, and the base peak of the spectrum
8
100-
I-
H2N-CH-COOH
I
s
116 I
S Z ' 3
NH-C-NHz
50-
1
I
--
NH
r
117
I
n
I1
412
I # (
I,
6.6. Analysis of Drugs and Drug Metabolites
Since 1971 the F D investigations of anti-cancer drugs have
played a major role in exploring the scope of F D mass spectroscopy[581.EI and F D spectra of propane- and butanesultams
(some ofwhich show an antitumor effect against Ehrlich ascites
tumors) were obtained; the F D spectra exhibit the following
five general phenomena:
1) High intensity of the molecular ions,
2) formation of most of the fragments in “one step”,
3) elimination of neutral particles (HzO, NH3, HCOOH)
from the protonated molecule,
4) formation of fragments by direct bond fission, and
5) production of doubly charged ions.
The extension of studies on model compounds, such as
e. g. medicinal car barn ate^[^^], to the investigation of cyclophosphamide (1 ) (Endoxan)and its metabolites[59]represents
an important advance in the direction of solving “real” analytical problems by field desorption. According to these investigations, individual components can be identified by means of
high-resolution F D spectra not only in mixtures of the parent
compounds themselves (as in Section 6.2) but also in mixtures
of parent compounds with metabolites. Fig. 17 shows a part
of the F D spectrum of a mixture of cyclophosphamide (1)
with three of its most important metabolites ( 2 ) , ( 3 ) , and
( 4 ) ; although the polarities of these compounds extend over
a wide range-(4) was stabilized as its cyclohexylammonium
salt-the molecular ions of all the components appear with
high relative intensities. The degradation products of cyclophosphamide could also be demonstrated (after thin-layer
chromatography) in extracts of body fluids. However, the
FD spectra were “electron-impact-like” and rich in lines
because of the impurities carried over, and interpretation of
these spectra is very difficult without additional information
about the compounds looked for.
mle
-
Fig. 18 a ) El mass spectrum of azathioprine (Imuran). electron energy 70 eV:
b) F D mass spectrum ofazathioprine, emitter heating current T* = 25 mA [60].
measurements by EI and by FD. Greater importance in this
connection attaches to a metabolite of azathioprine, namely
aglutathionyl conjugate; the EI spectrum in Fig. 19a is characterized predominantly by pyrolytic decomposition of the molecule. At T* (24mA) the F D spectrum once again yields exclusively the (M 1) ion. Above T* fragmentation can be induced
within a very narrow range of emitter heating currents (Fig.
19bl
+
I
HOOC -YH-CH2-CH2-C -NI
NH2
s
80150
200
-
Mo
250
mle
150
‘Lxl
60
~
Fig. 17. F D mass spectrum of a mixture of cyclophosphamide ( 1 ) and
three of its important metabolites ( 2 ) . (3). and (4) [59].
20
Azathioprine (Imuran) is a widely used immunosuppressive
drug. The EI spectrum of this compound is shown in Fig.
18a[601;the relative intensity of the molecular ion is less
than 1 %. The F D spectrum at T* (22mA) shows exclusively
the (M + 1) ion. The use of heating currents above T* yields
the F D spectrum shown in Fig. 18b; it is clear that the
signals in the higher-mass range of the EI and of the F D
(at 25 mA) spectrum are due to the same fragments (e.g. m/e
231, 152, 119). Apparently here the predominant thermal
decomposition of the azathioprine molecule in EI is simulated
by the thermally-/field-induced fragmentation in FD; the same
elemental composition of these ions is found in high-resolution
Angew.
Chrm. iiirrrnat. Edit.
Vol. 1 4 ( 1 9 7 5 )
I
No. 6
126
mle
-
Fig. 19. a) EI mass spectrum of l-methyl-5-nitro-5-(S-glutathionyl)imidazole,
electron energy 70eV [@I; b) F D mass spectrum of the same compound
at 27 mA emitter heating current ( > T * ) [60].
To explore the possibilities of field desorption in the detection of metabolites, a sample was isolated from the urine
of a rat to which azathioprine had been administered orally.
After column chromatography and purification by paper chromatography a cysteinyl conjugate could be identified in the
F D spectrumlhol.
413
7. Advantages and Disadvantages of the FD Method
in Comparison with Other Ionization Methods
Let us compare the F D method with other ionization
methods that are also regarded as “mild”, e . g . C1, FI, and
EI at low electron energies. The F D method is very suitable
for qualitative analysis of complex mixtures, but currently
still less suitable for quantitative analysis of such mixtures. AS
has already been explained in Section 4, there is an optimal
T* of the field anode for each component of the mixture.
At a constant anode temperature the F D signals are certainly
not proportional to the molar fractions of the components
of a mixture of substances in solution. Moreover, it is hardly
likely that it will be possible to obtain such proportionality
by introducing a variable temperature program. The intensities
of the ions from any one substance depend in a complicated
manner on the structure and the chemical properties of the
organic microneedles grown on the emitter surface, on the
concentrations of the individual components in the mixture,
on the thickness of the sample layer deposited on the microneedles, on the solvent, and on other parameters such as
the residence time of the sample on the field anode during
field desorption.
The potential of the F D method lies undoubtedly in the
field of qualitative analysis of substances that are thermally
labile on evaporation. Nevertheless, it must be emphasized
that the F D spectrum of a substance is, in the present state
of the technique, less reproducible than a CI or FI spectrum.
This aspect is, however, of minor importance since the F D
method is used in those cases where, as a result of their
low volatility, the samples do not afford unambiguously identifiable molecular ions in the CI and FI or the EI methods.
Under these circumstances combination of F D with EI and/or
CI is optimal. A great advantage of the F D method is that
the substances, which are often available only in picogram
amounts, do not need to be treated chemically in order to
increase their volatility. The major application of the F D
method is thus in theanalysis of very small quantities ofstrongly
polar, underiuatized substances.
An advantage of the electron-impact method is the wealth
of structurally specific fragments that are observed in the
mass spectrum. In the 30 or so years since the EI method
gained importance as an analytical tool experience has been
gained with a great number of substances, which has resulted
in extensive catalogs of spectra and many empirical rules
concerning fragmentation.
The method of chemical ionization introduced by Munson
and Field“’ has been used for only about four years in
a rather large number of laboratories; the F D method has
been tested and further developed systematically for only
about three years, mainly by the Bonn group. Nevertheless,
it is already recognized that there are many specific applications for which F D will prove invaluable.
8. Prospects
8.1. The FD Quadruple Mass Spectrometer
While F D quadrupole mass spectrometry is still in its
infancy, it could become important in special areas of F D
mass spectrometry. The significance of a rapid mass scan
414
has already been mentioned in Section 5 for the case where
electrical ion recording is used.
The quadrupole mass spectrometer (QMS) can be used
for the F D technique at low resolution ( < 5 0 0 ) because of
the following reasons[621:
1. The mass scan rate can reach the order of lo00 mass
units per second.
2. The intensity of the ion current and the resolving power
are almost independent of the energy of ions entering the
QMS (the maximal energy for EI is about 5-10eV).
3. The product of the transmission ( T ) and the resolving
power ( A ) is almost constant in the lower-mass region[631
(transmission is defined as the ratio of the current measured
at the first dynode of the SEV ion detector to the total ion
current emitted from the field anode). The ratio VA can be set
electronically on the QMS; thus, when desired, a large mass
range at large transmission and low resolving power can be
arranged, providing a rapid survey of the F D mass spectrum,
or the resolving power can be raised so that, for instance, the
molecular ion group can be analyzed more accurately.
The high recording rate with the QMS makes it possible
to adapt the processing of the F D mass spectrum to the
specific desorption behavior of the substance under analysis.
This is important for studies on the fundamental phenomena
of field desorption and also for the application of F D in
analysis. As an example we may mention the rapid determination of certain components in a mixture of substances such
as may be obtained in chemical synthesis of compounds or
by extraction methods in biochemistry[641.
8.2. Pyrolysis-MassSpectrometry
Some of the biologically most important active compounds
(polysaccharides, polynucleotides, and polypeptides) have very
high molecular weight (>20000). Controlled thermal degradation and identification of the structural units by mass-spectrometric methods holds out good prospects in two directions:
1. Rapid, accurate, and reproducible analysis of the pyrolysis
products, e.g. with a combination of Curie point pyrolyzer,
quadrupole MS, and data system, leads to fingerprint spectraC6’].The use of this system, with comparison of the spectra,
can provide unambiguous identification of known polymers.
2. Identification of ionized primary fragments, which should
be as large as possible, in the mass spectrum provides indications of the composition and structure of known and still
unknown polymers. Pyrolysis field desorption is suitable for
investigations on biopolymers[”*661 and industrial polymerization products, because of the very small consumption of
the sample material ( z10-sg), the very close sequence, both
in time and space, of pyrolysis and ionization, as well as
the mild ionization.
It must be emphasized that this promising and very recently
developed technique of combining pyrolysis and field desorp
tion can be applied only when two fundamental prerequisites
are satisfied. In the first place, very high demands are made
on the resolving power of the mass spectrometer employed.
Without excellent values for the resolving power (> 30000)
unequivocal assignment of the peaks runs into increasing
dificulties with increasing mass number. Secondly, the extremely complicated spectra of the multicomponent mixtures
encountered can be evaluated only if electronic data processing
is available.
Aiiqex.
Chmm. blrernar. Edir.
I
Val. 14 ( 1 9 7 5 ) ’ No. 6
8.3. Liquid Chromatography-Field Desorption
The coupling of a gas chromatograph to the mass spectrometer is regarded as the most successful mass-spectrometric
combination. However, compounds are amenable to this system only if they themselves are sufficiently volatile or if
this volatility can be produced by chemical pretreatment,
i.e. by the preparation of derivatives. In view of the rapid
progress in both high-pressure liquid chromatography and
field desorption, and in order to avoid vaporization of the
analytical sample, a combination of the two methods appears
promising. The first experiments in this direction were made
by an off-line technique[671.The eluate from the liquid chromatograph was collected, concentrated on the emitter, and analyzed by FD spectrometry. In this sequence the FD mass
spectrometer serves as a (second) molecule-specific detector’68!
I t remains to be seen whether the great efforts devoted to
the on-line technique‘”] will lead to the solution of the enormous technical problems, especially as regards the analysis
of highly polar substances, and whether the analytical information obtained by this direct coupling will be justified in view
of the effort and expense involved.
We thank the Deutsche Forschungsgeiiieinschafr, the Landesuint , f i r Forschuiig des Landes Nordrhein- Westfalen and the
Fonds der Chemischen Industrie for generous financial support
of our work.
Received: November 25. 1975 [A 63 IE]
German version: Angew. Chem. 87, 425 (1975)
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415
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