GB2091937A - Mass analysis sample ionisation - Google Patents

Abstract

The ionisation, for mass spectrometric analysis of materials which are normally solid and cannot be vaporised in a stable form, including many compounds of bio- medical significance such as saccharides, peptides and antibiotics is effected by subjecting a mobile sample of the compound to bombardment by a beam of neutral atomic or molecular particles. A target coating is prepared in which a sample of the compound is present in a mobile condition, eg. in a molten state or in solution in a medium of low volatility such as glycerol, at the surface of the coating and the atom beam is directed at a small angle, typically from 10 to 30 DEG with respect to the surface. The kinetic energy of the beam is such that during mass analysis a range of ion species is observed which includes both pseudo-molecular ions and structurally significant fragment ions. As shown, primary argon ions, produced in gas-filled discharge chamber 21, are directed into an argon-filled chamber 30 where resonant charge exchange occurs to provide a beam of neutral argon atoms, typically of energy 2-6 kev. The neutral beam is incident on the surface of target 38, which may be suitably heated or cooled and whose polarity determines whether positive or negative ions are selected for the slit-system of the mass analyzer, eg. of the double focussing type. <IMAGE>

Description

SPECIFICATION Investigation of molecular structure The invention relates to the investigation of the molecular structure of compounds and has particular reference to methods involving the ionisation of involatile or thermally unstable molecules of complex materials.

The instrumental technique involved in such methods is almost necessarily that of mass spectrometry and the invention utilises that technique. The analytical procedures of mass spectrometry are however generally well established and will not be discussed in any detail. The production of ions for observation in the spectrometer has also been extensively studied in the last twenty years and a number of standard techniques are available and are satisfactory for many materials. The problem remains that particularly for involatile materials (those which cannot be evaporated without change of structure), among which are compounds of biomedical significance such as saccharides, peptides and glycosidic antibiotics, no completely satisfactory method of ionisation has been found.

Conventional methods of ionisation such as electron impact, chemi-ionisation and field ionisation require the sample to be vaporised.

Involatile materials are therefore excluded and thermally unstable molecules may be decomposed. Even for suitable materials the analytical information which can be obtained is limited. In using electron impact, energy in excess of that required for ionisation is transferred to the ion which may then form fragment ions. This process may occur so rapidly that little or none of the parent ion is detectable in the spectrometer and no genetic relationship can be established between fragments and parent. Conversely the so-called soft ionisation techniques of chemi-ionisation and field ionisation involve such little energy transfer that only the parent ion may be observed when fragmentation products would be of interest.

A further soft ionisation technique, that of field desorption, which is available for use with a solid sample, gives useful results for compounds of low volatility many of which are thermally unstable. Again however, fragmentation occurs infrequently so that little structural information is obtained and data is largely confined to the parent ion. Furthermore the technique employs a sample deposited in minute quantity on a fragile activated ion emitter which is difficult to prepare and may have a short useful life.

As indicated in the recent book "Mass Spectrometry", edited by Merritt and McEwan and published by Marcel Dekker Inc. (1979), the ionisation methods noted in the preceding paragraphs remain standard techniques in the analytical chemistry of organic substances; of these techniques field desorption is the only standard procedure for labile organic compounds in the liquid and solid phases. The book also refers to recent reports on the use of positive ion bombardment as a means of ionisation but, in the experience of the present inventors, that technique presents a number of difficulties. These include the accumulation of charge on the surface of any sample material having insulating properties, so that the observation period is very restricted.

It is an object of the invention to provide a method suitable for investigation of materials hitherto regarded as intractable, in which a range of related ion species is observable for structural determination over a relatively extended period.

In accordance with the present invention there is provided a method for the investigation of the molecular structure of a normally solid involatile compound, the method comprising the operations of forming on a target a coating in which a sample of the compound is present in a mobile condition at the exposed surface of the coating, bombarding said surface with electrically neutral molecular particles which are incident at an angle with said surface not exceeding 45" and have kinetic energy such that the overall result of the bombardment is the production of a range of ion species, including both pseudo-molecular ions and structurally significant fragment ions, and subjecting said range of ion species to mass analysis.

With a method according to the invention the period during which the ion spectrum may be observed is found to be prolonged compared with that obtained on similar bombardment of a solid sample of the same compound. This result is thought to be attributable to a capability for continuous replenishment of the compound at the bombarded surface of the coating.

The coating may consist of the compound to be investigated, maintained in a molten state by heating, but more commonly it will be appropriate for the compound to be rendered in a mobile condition by solution or suspension in a suitable medium.

For a sample in the dissolved state the medium must be a solvent of low volatility since it is to be used under vacuum and may be a higher glycol such as glycerol or another material of comparable physical properties.

The concentration of sample material in the medium and the total quantity of the sample are parameters which determine the lower limit of sensitivity of ion detection and thence the degree of structural information which may be derived from the mass spectrum. The concentration may extend over the range from 10-6 Mol. dm -3 to 1 Mol. dm-3 and the minimum sample quantity may be in the order of 0.1 gm.

The molecular particles for bombardment are conveniently derived from an ionised rare gas and are then usually atomic. The nature of the ionisation process caused by such atomic bombardment is difficult to analyse but it is useful to compare the products with those of the standard techniques. It will be recalled that electron impact ionisation (El) and chemical ionisation (Cl) tend to be complementary in that El gives structural information as a result of fragmentation while Cl gives only molecular mass information because there is very little fragmentation. This difference in behaviour is partly a question of the internal energy distribution of the ions (broad for El; very narrow for Cl) but also derives from a tendency for El to produce ion radicals (ions having an odd number of electrons) while CI produces even electron species which are inherently more stable.Such even electron species are most commonly displaced by + 1 atomic mass unit from the true molecular weight of the sample material but of course continue to be precise indicators of that weight. The term 'molecular ion' refers to an ion whose mass: charge ratio m/z is equivalent to the molecular weight of the compound.

It is convenient therefor to describe an ion which is a known modification of the molecular ion as a 'pseudo-molecular' ion. When the modification represents the gain or loss of a proton or hydride ion the possible species are denoted by [M + HJ+ and (M - HJ+ for the positive ions and by [M - H]- and [M + H]for the negative ions.

It is now observed as a generalisation that in the spectra obtained by the method of the invention the parent molecule is represented by the pseudo-molecular ion. The presence of a broad band of ion energies characteristic of a bombardment process ensures that fragmentation still occurs to some degree so that structural information is available while sufficient ions of the relatively stable, even electron species persist to give the parent mass information. Thus the desirable features of electron impact ionisation and of chemical ionisation are obtained in a single experiment.

The temperature of the target may be controlled by heating or cooling in order to maintain the sample in the desired condition.

For suitable sample materials it is advantageous to provide means whereby a pseudo-molecular ion of even electron species is produced by the attachment to the sample compound of a cation other than H + such as an alkali metal ion or an anion such as a halide ion. The effect is to modify the extent and mode of fragmentation process so that a different range of information is obtained in the spectrometer. This modification may be achieved by converting the sample into an appropriate salt or by adding suitable ionic compounds.

The angle between the direction of incidence of the particles and the surface of the coating is preferably within the range 10 30 . As the angle of bombardment is increased the probability increases that the desired representative pseudo-molecular ions will not be produced intact at the surface but that only fragment ions will result. If the angle is made very small a higher beam energy may be tolerated but the interaction with the surface material becomes increasingly difficult to control.

The neutral particles may be produced by charge neutralisation from a beam of ions having an energy which will generally not exceed 10 keV. The range from 2 keV to 6 keV produces an adequate yield of pseudomolecular ions from many materials of interest.

The full exploitation of the method may require the use of a refined design of mass analyser such as a high resolution doublefocusing instrument for deriving information about the structure of a molecule by means of the genetic relationship of its fragmentation products. It is also an advantage of the method that a quadrupole filter or singlefocusing spectrometer may now be used in the classical mode to derive simple structures or to confirm the presence of a suspected molecule in a material hitherto regarded as intractable.

The nature of the invention will be further made clear by describing an embodiment of suitable apparatus and the manner of operation with reference to the accompanying drawings in which: Figure 1 represents schematically a mass spectrometer having double focusing geometry; Figure 2 represents diagrammatically an ion source for a mass spectrometer for use in accordance with the invention; Figures 3 to 7 represent mass spectra obtained in accordance with the invention; and Figure 8 represents diagrammatically the fragmentation scheme derived from the spectra of Figs. 4 to 7.

A mass spectrometer essentially comprises an ionisation source to derive ions from a sample for admission to a mass analyser and before considering the ion source in detail a conventional form of the mass analyser will be briefly reviewed. A substantially mono-energetic beam of a single ion species entering a uniform magnetic field is deflected in a circular arc, the radius being dependent on the mass-to-charge ratio of the ion. Ions of different mass-to-charge ratios in a mixed beam may therefore be separately detected in their respective orbits. Alternatively each species may be brought in turn into the same orbit by scanning through a range of accelerating voltages at the beam input or by scanning through a range of values of magnetic field.

Such orbits are accommodated between polepieces which generally are sectors of a circle.

The single-focusing mass spectrometer, as described in principle in the preceding paragraph discriminates between particles according to their momentum. Poor resolution of each mass value is obtained therefore when any significant spread of initial velocity is present. Since ions produced by bombardment of a surface have a characteristically broad energy distribution, mass analysis of such ions will usually require the use of a double focusing technique which provides further resolution. This is achieved by applying an electrostatic deflection field, usually in advance of the magnetic analyser, to select from or to focus the initial beam in response to the kinetic energies of the particles.

Fig. 1 shows schematically a mass analyser embodying double-focusing in the configuration known as Nier-Johnson geometry. An ion beam 10 is produced by bombardment at a target (not shown). The beam width is limited by a slit system 11 for admission to the annular gap between two electrostatic deflection plates 1 2 and 1 3 which comprise respectively sector portions of outer and inner concentric cylindrical surfaces. The potential at slit 11 is the same as the mean potential (typically zero) of the plates 1 2, 1 3 which are symmetrically energised so that the beam enters the plate-gap from a field-free region.

On entry the beam is inevitably divergent to some extent and also, as a previously noted characteristic of bombardment ionisation, comprises ions having a broad distribution of energies. The effect of the radial electrostatic field in the plate-gap is to separate the ion trajectories according to energy but also to converge those ions of the same energy which initially followed divergent paths. The resultant direction focusing is indicated at a plane 1 5 beyond the plates 12, 1 3. The beam continues through a second field-free region to enter a magnetic field sector 1 6 which as viewed, produces a field perpendicular to the plane of the paper. Separation of the component particles of the beam into respective circular orbits then occurs according to the mass:charge ratio.Focusing also occurs so that on exit from the sector 1 6 a point can be found for each species at which direction and velocity focusing coincide. The double-focusing point for a single mass:charge ratio is.

indicated at 1 7. An electron multiplier 1 8 receives the beam at point 1 7 and is connected to conventional current measurement and data recording/processing apparatus (not shown). In the mass spectrometer other species are brought in turn to the same doublefocusing point by sweeping through a range of values of magnetic field. Alternatively, for mass spectrography the mass separated ions would be detected simultaneously by means of a photographic plate or an electron multiplier array.

Fig. 2 shows in cross-sectional view the arrangement of a source 1 9 which provides ions to be analysed (conveniently referred to for the purpose of this specification as 'analytical ions') for input to the slit system 11 of Fig. 1. Analytical ions are produced in the source 1 9 by bombardment of a sample with neutral atoms which are derived by charge exchange from a beam of positive ions, to be referred to herein as 'primary ions'. For simplicity the whole of the source 1 9 including a sample chamber is indicated as forming a single connected vacuum enclosure. In practice since the various stages of ion production have different vacuum requirements the enclosure will comprise compartments coupled by standard flange fittings and with the appropriate high vacuum or gas leakage connections to each compartment.Such structural components are in common use and will not be shown.

As a first stage in the production of analytical ions the source 1 9 includes on an axis 20 a cylindrical gas-filled discharge chamber 21 for the generation of primary ions. A cylindrical anode 22 mounted concentrically within chamber 21 is maintained at a high positive voltage such that ionisation occurs in an argon atmosphere at about 10-3 torr. The efficiency of ionisation can be increased by providing a magnetic field by means of a solenoid 23 mounted coaxially with chamber 21 and externally to it. Primary ions are extracted from chamber 21 at an axial aperture 21A with an energy typically in the range 2 to 6 keV, and formed into a beam in a high vacuum region containing a three-element electrostatic lens 24.The beam is additionally controlled in direction by pairs of electrostatic deflection plates 25, 26 for passage through a pair of apertures 27, 28 spaced apart along the axis 20. Between the apertures 27, 28 the beam enters a chamber 30 which confines an atmosphere of argon maintained at a pressure of 10-4 torr. Within chamber 30 resonant charge exchange occurs to provide a beam of argon atoms along the axis 20 having nearly the same energy as the primary ion beam.

Charge is dissipated by transfer to the wall of chamber 30 which is at earth potential. Some primary ions continue to travel with the atomic beam and are deflected away from axis 20 by electrostatic deflection plates 31. Beam current sampling grids 32 are placed immediately after the apertures 27, 28 for monitoring the efficiency of charge exchange in chamber 30. Typically, the density of the neutral beam is in the range from 1013 to 1011 atoms/ cm2/sec. After passing the plates 31 the atomic beam enters a flight tube 33 leading to a sample chamber 34 arranged for mounting on a conventional double-focusing mass spectrometer such as the AEI type MS902.

The mounting axis of chamber 34 is perpendicular to axis 20 and is the normal ion admission axis 35, via slit-system 11 to the mass analyser. A sample probe 36 is arranged for entry to chamber 34 along axis 35 through a standard vacuum lock 37. Probe 36 is terminated in a metallic (usually copper) sample carrier or target 38 having a face which can be set at an angle up to 45" to the axis 20 and can be heated or cooled. Target 38 can be formed as a block having internal passages 39 through which a heating fluid or a cooling fluid such as liquid nitrogen can be pumped. In an alternative form target 38 comprises a thin sheet of metal to which electrical connections (not shown) can be made for resistive heating.The compound to be investigated is prepared in a thin liquid coating on the inclined face of target 38 such that under atomic bombardment the analytical ions produced are readily attracted through the slit-system 11. Probe 36 can be steered in three dimensions by micrometer drives (not shown) situated outside the vacuum lock, to expose any desired part of the sample to the atomic beam. Additionally the target 38 is electrically biased relative to slit-system 11 so that analytical ions enter the mass analyser with the desired mean energy. A bias supply up to + 8 kV has been used and the high field-strength inherent in this arrangement results in ion extraction efficiencies much higher than those obtained previously.The polarity of target 38 relative to slit-system 11 is chosen to select either positive or negative ions from the sputtered particles, without affecting the production of the atom beam.

It is clear that the sample chamber 34 may be adapted to accommodate the conventional ionisation techniques in addition to the atombeam forming structure so that alternative methods may be used without demounting the apparatus.

It will also be appreciated that within the scope of the invention the beam of atomic or other neutral particles may be produced in any suitable manner and particularly that forms of primary ion source and charge exchange system other than those described with reference to Fig. 2 may be used.

In describing the apparatus of Fig. 2 only brief reference has been made to some aspects of the experimental procedure which are important in obtaining the results to be described later. Thus the energy of the primary ion beam, which is substantially preserved in the subsequently neutralised beam, is indicated as typically within the range from 2 to 6 keV but this is not a critical choice. In general a larger value will be appropriate to a small angle of approach. A much lower value will generate an adequate number of analytical ions provided that the sputter ion energy threshold, typically about 20 eV, is exceeded but the primary beam becomes more difficult to focus and direct in the presence of extraneous fields.At a value much higher than, say, 10 keV the beam becomes increasingly destructive and uncertainty is then introduced as to whether the analytical ions extracted will provide the desired structural information. It is emphasised that the purpose in carrying out the present invention is to derive both pseudo molecular ions and structurally significant fragment ions which together provide such information.

A more significant factor in achieving this purpose is the choice of the angle with respect to the surface at which the neutral beam is arranged to strike the target coating. A range up to 45 is available in the adjustment of the face of target 38 in Fig. 2 but an angle no greater than 30 is preferred and 20 has been found to be suitable in the investigations so far carried out. If the angle is increased to 45 or above it becomes increasingly probable that no intact molecule will escape from the surface and that disintegration will occur at the point of bombardment.The angle may be reduced below 20 but an excessive reduction will progressively introduce an undesirable uncertainty in defining the geometry of the impact area and a consistent energy interaction with the surface will be increasingly difficult to maintain. An angle of about 10 is thought to be the lower limit of usefulness.

The preparation of samples is very simple but has proved to be of great significance in obtaining persistent spectra. The target surface (the substrate) is cleaned and may be etched chemically using acid or mechanically roughened to improve surface contact. In early experiments spectra were obtained for materials such as peptides and saccharides which had been deposited on the substrate from aqueous solution and then dried. It has been observed however that for solid or dry-deposit samples the ion spectrum is often transient, the molecular ion of interest fading in intensity during a period of a few minutes. The cause of the fading is not clear but possibly contributory causes are surface damage by the atom beam and surface contamination by residual gases.Bombardment damage can be limited by restricting the energy or the intensity of the beam and its angle of impact at the surface and also by using a large sample area (0.5 cm2 or greater) which is traversed through the impact point. The rate of contamination is acceptably low only if the pressure in the sample region of the spectrometer is held at or below 10 - 8T but it is appreciated that this will not be achieved in most large instruments. It has however proved practicable in accordance with the invention to obtain spectra with no depletion of parent ion sensitivity over periods of hours by solution of sample compounds in suitable media of low volatility.

Such stability in the behaviour of the sample leads to the belief that continuous replenishment of the compound occurs at the bombarded surface of the coating when the sample is prepared in a mobile condition.

The involatile materials of interest, particu larly in a bio-medical context, are generally soluble in water and in other solvents of high polarity and the principal criterion of selection of a sample medium is that of low volatility.

Glycerol has been used in the present work but other polyhydric systems would be suitable. In some cases the natural solubility may be too low and can be improved by the use of a wetting agent. For example in an experiment on chlorophyll-A solubility in glycerol was found to be very poor and the sample would not yield a spectrum. The addition of 1 % of the agent known commercially as TRI TON-X100 to the glycerol produced a solution from which a full spectrum was obtained.

The relatively high viscosity of glycerol is of possible advantage in restricting flow on the target surface but the quantity of coating is generally so small that bulk flow is not a serious consideration. The range of concentration of sample material in the medium which may be employed extends at least from 10-6 Mol. dm-3 to 1 Mol. dm-3. For maximum sensitivity the bulk concentration is sufficient, it is believed, to maintain a surface concentration of sample molecules equivalent to a continuous monolayer. In this condition the medium material appears in the spectrum at very low intensity or not at all.In volume of solution of, say, 10y1 the quantity of solid sample material at mass 10duo could range from O.01gm to 0.O1gm. The smaller quantity would yield a spectrum containing only the most intense peak or peaks but it is an advantage of the method that an indication of any kind should be obtainable from sample sizes in the order of tens of nanograms. It is of course preferable to increase the sample size towards the upper end of the range at which a complete spectrum, including fragments at low intensity, will be obtained.

It has been further appreciated by the present inventors that the scope of the experiment can be broadened and the fragmentation pathways modified by introducing stablising anions, or cations other than H +, into the pseudo-molecular ion. This process may arise from the prepared form of the sample which is chosen or may be induced. For example spectra have been obtained from commercially formulated salts of antibiotics such as that shown in Fig. 3 for neomycin sulphate which in this form has proved almost impossible to characterise by any other method of mass spectrometry. In other cases it is a simple matter to add a salt to the solution or suspension containing the sample.For example NaCI, KBror NH4CI will prouduce respectively the stabilised positive ions [M + Na]+, [M + K]+ or [M + NH]4+ and the corresponding negative ions [M + Cl]-, [M + Br]- or [M + Cl]-.

The information to be derived can be selected by this means to the extent that an increase in stability increases the pseudo-molecular ion sensitivity but reduces the fragmentation data.

In some cases, such as a sugar having an attached ammonium ion, the fragmentation scheme may be altered.

Some further experimental results will be described in outline in order to demonstrate the application of atomic bombardment in essentially new areas of investigation. It has been possible for example to demonstrate for the first time the use of mass measurement of high accuracy to establish unambiguous atomic compositions for sputtered molecular ions. For the monosaccharide ribose the fragment ions at m/z = 133, 73 and 45 have been shown to have compositions: C5H9O4+, C3H5O2+ and C2H50+ respectively.

Additionally, the fragmentation pathways between these fragment ions have been established by the detection of the corresponding metastable transitions,

Such transitions are observed in the first fieldfree region (as it is conventionally known) which lies between the exist slit of the ion source and the entrance to the electrostatic analyser. The demonstration of the existence of such transitions for sputtered ions, not previously observed, is expected to advance the understanding of the sputtering process and of surface reactions, in addition to broadening the scope of molecular structure determination.

As a further example of the ability to produce detailed information concerning molecular structure, the results obtained for the tripeptide Alanyl-leucyl-glycine will now be described with reference to Figs. 4 to 8.

The distinction between the results observed according to the sign of the analytical ion obtained by atom bombardment of the tripeptide is shown in Figs. 4 and 5. Fig. 4 shows the positive ion spectrum while Fig. 5 is the corresponding negative ion spectrum. Both show good pseudo-molecular ion intensity, the former at m/z 260 corresponding to [M + H]+, the latter at m/z 258 corresponding to [M - H]-, where M is the neutral molecule.

Using a mass spectrometer such as the AEI MS902 it is possible to observe metastable peaks at non-integral mass numbers which correspond to transitions occurring in the second field-free region of the spectrometer, which region lies between the exit from the electrostatic analyser and the entrance to the magnetic sector. As shown in Fig. 6 a metastable peak occurs at m/z 131.6 (= 1 852/260) due to the transition:

Additionally, the precursor ions of an ob served fragment ion may be determined by the detection of metastable transitions occuring in the first-field-free region.Fig. 7 shows the results of such an experiment where, by scanning through a range of ion acceleration voltage V, the precursors of the fragment ion at m/z 185 are determined to be the ions at m/z 203, 215, 242 and 260, the latter being the pseudo-molecular ion.

By the application of these methods a full fragmentation scheme may be determined as.

shown in Fig. 8, from which the molecular structure of this tripeptide can be unambiguously deduced.

The method has also been shown to be applicable to molecules of particularly high molecular weight such as the undecapeptidc, methionyl-lysyl-bradykinin of mass 1318.

Such results have shown that the use of a atomic beam ion source with a doublefocus- ing mass spectrometer enables high mass resolving power and the detection of metastable transitions to be achieved. However the Nier-Johnson geometry of the analyser of Fig.

1, although it gives first and second order accuracy in direction focusing, does not give the best available degree of energy focusing and it is probable that further improved mass resolution and sensitivity can be achieved with instruments having better energy focusing.

Other geometries can also be used in doublefocusing instruments, following known analytical procedures and auxiliary means of inducing fragmentation can be introduced, such as a gas-filled collision cell placed in a field-free region.

Nevertheless a simpler spectrometer such as a quadrupole or a single-focusing magnetic instrument may be used in accordance with the invention. Spectra of lower quality are obtained, particularly in respect of mass resolution, and the products of metastable fragmentation cannot be genetically related, but within these constraints there are useful analytical applications.

The use of the method of the invention has been explained in providing a capability for the structural investigation of many involatile solid compounds of industrial and bio-medical significance which have previously been considered intractable subjects for mass spectrometric observation. One result of the method is that the wide energy distribution among the ionisation products of a bombardment process can be used to advantage, particularly by means of the double-focusing spectrometer, the observation of both pseudo-molecular ions and metastable transitions.

Claims (7)

1. A method for the investigation of the molecular structure of a normally solid involatile compound, the method comprising the operations of forming on a target a coating in which a sample of the compound is present Ir.

a mobile condition at the exposed surface of the coating, bombarding said surface with electrically neutral molecular particles which are incident at an angle with said surface not exceeding 45 and have kinetic energy such that the overall result of the bombardment is the production of a range of ion species including both pseudo-molecular ions and structurally significant fragment ions and subjecting said range of ion species to mass analysis.

2. A method according to Claim 1 in which the sample compound is dissolved in a medium of low volatility.

3. A method according to Claim 2 in which the medium is a glycol.

4. A method according to Claim 3 in which the medium is glycerol.

5. A method according to any preceding claim in which the sample is chemically modified before bombardment such that the psuedo-molecular ions resulting from bombardment contain a predetermined stabilising ion other than a proton.

6. A method according to any preceding claim in which the surface of the coating is bombarded with particles which are incident at an angle with said surface in the range 10 to 30 .

7. A method according to Claim 6 in which the kinetic energy of the particles has a value within the range 2 keV to 6 keV.