AU599149B2 - Electron spin resonance spectrometer - Google Patents

Description

i i-;I AU-AI-79187/87 PCT WORLD INTELLECTUAL PROPERTY ORGANIZATION INTERNATIONAL APPLICATION PU4 H U RT TE P 4 ,N1 OOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 01984 C01N 24/10 Al (43) International Publication Date: 24 March 1988 (24.03.88) (21) International Application Number: PCT/AU87/00296 Canterbury, VIC 3126 McLAREN, Neil, R. 11/32 Marne Street, South Yarra, VIC 3141 (AU).

(22) International Filing Date: 28 August 1987 (28.08.87) (74) Agent: CLEMENT HACK CO.; 601 St. Kilda Road, Melbourne, VIC 3004 (AU), (31) Priority Application Numbers: PH 8127 PI 3019 (81) Designated States: AT (European patent), AU, BE (Eu- (32) Priority Dates: 19 September 1986 (19,09.86) ropean patent), CH (European patent), DE (Euro- 9 July 1987 (09.07.87) pean patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European pa- (33) Priority Country: AU tent), NL (European patent), SE (European patent).

(71) Applicants: MONASH UNIVERSITY [AU/AU]; Wel- Published lington Road, Clayton, VIC 3168 ROYAL With international search report, MELBOURNE INSTITUTE OF TECHNOLOGY [AU/AU]; 124 La Trobe Street, Melbourne, VIC 3000 A J, P.

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(72) Inventors: PILBROW, John, R. 12 Elliott Crescent, AUSTRLAN Dingley, VIC 3172 TROUP,, Gordon, F.

132 Canterbury Road, Canterbury, VIC 3126 (AU. -7 APR 1988 TIRKEL, Anatol, Z, 21 Walstrab Street, East Brighton, VIC 3137 HUTTON, Donald, R, 30 PATENT OiCE Guest Road, South Oakleigh, VIC 3167 GRUN- ER, Lucian 3 Mont Albert Road, (54) Title: ELECTRON SPIN RESONANCE SPECTROMETER 36 38 14/ 20210 22 32 (57) Abstract An electron spin resonance spectrometer which includes a sa.Tiple arm (16) and a reference arm (18) The sample arm has a circulator (30) for directing radiation to a resonator (22) in which a sample for analysis is deposited and, also for receiving radiation from the resonator (22) and directing it to a detector The detector (32) detects the microwave radi.

ation from the sample arm (16) and reference ari (18) to determine a property or properties of said sample. The sample arm (16) and said reference arm (18) are formed as an integrated microwave bridge which includes a divider (14) having a first output portion coupled to an attenuator said attenuator being coupled to said circulator (30) by a microwave transmission line. The resonator (22) is surrounded by a magnet (24) and modulation coils (26).

L u _i WO 88/01984 WO 8801984PCT/AU87/00296 1- ELECTRON SPI"' AESONANCE SPECTROMETER This invention real.tes to an electron spin resonance spectrometer.

Electron Spin Resonance (ESR) 8ometimes called Electron Paramagnetic Resonance (EPR) t is the resonant absorption of microwave radiation by a paramnagnetic system placed in a magnetic field.

Applications of ESR are to be found in physics (metal ions in crystals and powders), chemistry (crystals, chemical complexes in solution, free radicals and chemical reactions), biochemistry (particularly hente proteins, rion-heme iron proteins, copper proteins, th~e use of metal substitution such as cobalt in place of zinc in zinc enzymes and by means L- WO 88/01984 2 PCT/AU87/00296 of spin labelling of biological molecules including proteins and membrane components), natural materials (naturally occurring radicals and trace metal ions in coal and metal complexes in petroleum), gemmology distinguishing between natural and synthetic sapphires using a method developed by Drs. Hutton and Troup, co-inventors of the present invention), glass technology (magnetic ions in glasses and structural disorder). Other areas in which considerable development is possible include clinical medicine, testing for production of free radicals in gamma-irradiated food and in archaeometry.

Electron Spin Resonance (ESR), often called Electron Paramagnetic Resonance (EPR), is most often carried out at a fixed microwave frequency 9 to 10 GHz and spectra recorded as a function of an applied magnetic field. The microwave circuit is usually built around waveguide components and is therefore bulky. Research spectrometers have magnets weighing anything from 1 tonne to 3 tonne. Thus ESR spectometers are not readily portable. Even the smaller bench-top models which have been commercially available for 15 to 20 %ears are too heavy to be moved about.

It has recently been recoqnised that low microwave frequencies (1 to 4 GHz) provide improved resolution of spectra due to transition ions such as copper cobalt (II) and molybdenum as well as for samples of biological interest. Applications of low microwave frequencies (1 to 4 GHz) to ESR have benefited from a rediscovery in 1981 of an old principle of the side resonators which were incorporated in magnetron design at least as early as 1941 (Collins MIT Radiation Laboratory Series Volume 6 (1948)) and now referred to in the ESR literature as split-ring or looo-gap resonators, Which take the place of resonant sample cavities. In its simplest form such a resonator is a conducting cylinder with a capacitive slit along one side. By means of such resonators, sample volumes can be maintained at the levels presently used at 9 GHz with perhaps 2 to 3 times lower sensitivity. This makes ESR at 1 to 4 GHz a feasible proposition on a routine WO 88/01984 3 PCT/AU87/00296 basis. Loop-gap resonators constructed from a machinable ceramic (MACOR, made by Corning Glass Co.) and then electroplated, are easily made for use in the 2 to 4 GHz range.

The proposed microwave bridge and ESR spectrometer is not restricted to operation with the described resonators but may be used with any suitable sample resonant microwave structure.

The object of this invention is to provide a relatively low cost portable spectrometer which can operate somewhere in the 2 to 3 GHz range.

The invention provides an electron spin resonance spectrometer having a microwave source for. providing microwave radiation to a sample and a reference arm, said sample arm having a circulator for directing radiation to a resonator in which a sample for analysis is deposited and for receiving radiation from the resonator and directing it to a detector, said detector detecting said microwave radiation from the sample and reference arms to determine a property or propertes of said sample, characterised in that said sample arm and said reference arm are formed as an integrated microwave bridge.

Since the components are formed as an integrated microwave bridge the spectrometer will be inexpensive and the components can be incorporated to thereby provide a spectrometer which is light and therefore easily portable and which is capable of operating in the 2 to 3 GHz range.

Preferably the spectrometer includes an isolator for receiving radiation from the microwave source to prevent reflected radiation from returning to the source, a divider for directing some of said radiation to the sample and reference arms, an attenuator arranged in the sample arm between the divider and the circulator; a phase shifter and an attenuator arranged in the reference arm between the divider and the detector. A single ended or balanced mixer can be connected between the sample and reference arms as the detector.

WO 88/01984 4 PCT/AU87/00296 A preferred embodiment of the invention will be described in more detail with reference to the accompanying drawings in which: Figure 1 is a block diagram of embodi'ents of the invention; Figure 2 is a plan view of part of the ESR spectrometer of Figure 1; Figure 3 is a view showing the part of Figure 1 in the form of an electrical circuit diagram together with the remainder of the ESR spectrometer; Figure 4 is a cross-sectional side view along the line 5-5 in Figure 3; Figure 5 is a flow sheet showing computer control of the ESR spectrometer; and Figure 6 is a typical output of an ESR spectrometer.

With refeZence to Figure 1 a microwave source which may be a transistor oscillator mounted in a tunable resonant cavity or a prograrmable synthesizer chip using a suitable VCO (voltage controlled oscillator) digital frequency divider and phase lccked loop, prod.uces microwave radiation in about the 2 to 3 GHz range. That radiation is received by an isolator 12 which prevents reflected radiation passing back to the microwave source 10. The isolator 12 passes the radiation to a coupler 14 which splits the radiation such that some of the radiation is directed to a sample arm 16 and a reference arm 18. The sample arm 16 includes an attenuator 20. The attenuator is adjustable to alter the amount of microwave power falling on a sample provided in a resonator 22 which is surrounded by a magnet 24 and modulation coils 26. A circulator 30 receives the radiation from the attenuator and directs the radiation to the loop gap resonator 22 where a sample (not shown) is exposed to the radiation. The encoded radiation is then received by the circulator 30 which directs te radiation to a mixer 32.

The reference arm 18 includes a phase shifter 36 for variable phase shift control of the radiation in the reference arm and an attenuator 38. The phase shifter 36 and the

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WO088/01984 2/8116 I6 PC 5attenuator 20 may receivo an electronic control signal on lines 40 from a controller operated by a microprocessor or other computing apparatus. Similarly, the attenuator 38 may also receive an electronic control signal.

In a first embodiment of the invention the microwave radiation in the reference arm '18 is directed from the.

attenuator 38 to the mixer 32 on line 42 where it is combined with the radiation received by the circulator 30 from the resonator 22. The output signal from the mixer is a modulated wave form which can be processed by suitable electronic detection circuitry (not shown') to provide an output which can be interpreted. Generally all power in the sample arm is absorbed by the resonator 22 and the sample and reference arms are required to be balanced initially. When a paramagnetic resonance occurs, the bridge becomes unbalanced and the signal is received by input of mixer 32. The reference arm 18 enables the mixer 32 to operate at a suitable level. That radiation is combined with the radiation received from the resonator 22 to effectively maximise the, sensitivity of the system.

The isolator 12 and circulator 30 may be two port and three port ferrite components respectively which are constructed using stripline or microstrip techniques. The phase shifter 36 is preferably electronically controlled.

The resonator 22 is well known from scientific literature and therefore full details will not be described herein.

F'or general use the magnet 24 may be air-cored Helmholz pair magnet capable of linear sweeping uip to magnetic fields of about 0.15 Tesla which is approximately twice the field required to observe ESR from free electrons (87 mT at a microwave frequency of 2.5 GHZ) or a small swept electromagnet when portability is not of prime concern. For particular application to radicals or nitroxide spin labels the magnet 24 is preferably a permanent magnet based on Sm-Co alloys) whose field is set to a suitable value below the free electron resonance field and which can be swept above

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WO 88/01984 6 PCT/AU87/00296 this value by means of auxiliary coils 26. Such sweeping is preferably under microprocessor or computer control. In whichever magnet embodiment is used the magnetic field will be modulated at a suitable frequency probably not exceeding 100kHz and to detect at that frequency or some harmonic of that frequency to produce a spectral display. Such coils could consist of approximately 30 turni of 10"/1000 gauge copper wire. For homogeneity two coils are needed at the Helmholtz condition.

All of the components detailed in Figure 1 apart from the resonator 22, magnet and coils 24 and 26, can be integrated into a microwave bridge.

The preferred embodiment of the invention, is described in greater detail with reference to Figures 2 and 3.

Input port 70 receives microwave radiation from the microwave source 10 (Figure 1) and is a SMA connector which is coupled to one of the terminals of component 12. The component 12 is preferably a drop-in type circulator which is of conventional design'and therefore will not be described in further detail. The second terminal of the circulator is coupled to a Wilkinson divider 14 which performs the function of the coupler 14 described with reference to Figure 1. The third terminal of comonent 12 is provided with a 50 ohm termination 72 so that the circulator does in fact act as an isolator to prevent reflection of microwave energy from the Wilkinson divider 14 to the input port 70. Connected between the Wilkinson divider 14 and the isolator 12 is a DC decoupling capacitor 71.

The Wilkinson divider 14 splits the signal received from the second terminal of the isolator 12 so that microwave energy is directed to the reference arm 18 as well as the sample arm 16. The signal which is directed to the reference arm 18 is received by the phase shifter 36. The phase shifter 36 includes a circulator 73 which has a first terminal connected to one of the arms of the Wilkinson divider 14. The second terminal of the circulator 73 is connected to DC decoupling capacitor 74 which in turn is coupled to a i ii i WO 88/01984 PCT/AU87/00296 transmission line transformer 77 which in turn is coupled to a diode support 78 for supporting a parallel tuned varactor diode (not shown), and a tuner 79 for tuning the diode into parallel resonance. A configuration 75 which acts as an R.F.

choke is provided so that a D.C. bias signal can be applied to the diode circuit in such a way that the R.F. and D.C.

circuits are decoupled. The signal leaving port 2 of the circulator 73 encounters a reactive load consisting of the parallel tuned varactor diode which reflects said signal back to port 2 and hence to emerge at port 3 and be passed to line Said reactive load can be varied by the application of a suitable control voltage to input port 76 in order to adjust the phase of the signal appearing at terminal 3. The third terminal of the circulator 73 is coupled to microwave transmission line 80. The transmission line transformer 77 transforms the impedance of the line up to a suitable level for the most efficient operation of the diode.

The signal from line 80 is received by the attenuator 38 which includes a circulator 81 which has its second terminal coupled to PIN diode 82 via a DC decoupling capacitor 83 and microwave transmission line'84. The other end of microwave transmission line 84 is coupled to a 50 ohm termination 85 via DC decoupling capacitor 86. The microwave transmission line 84 is also coupled to a bias network 87 for providing an input current bias signal at port 88, By suitable application of the current signal from input port 88 to biasing network 87 via line 89 and to terminal 2 of circulator 81, the signal received at terminal 1 can be suitably attenuated and received at terminal 3 for supply to directional coupler 50. The directional coupler 50 is of the transmission line type, comprising a main transmission line ',0 and an auxiliary line 94 which is accessed by port 93 via transmission line 91 and terminated with a termination The transmission line directional coupler 50 makes it possible to divert a small fixed proportion of the input signal at port 93 for control purposes and monitoring. The transmission line is coupled to a hybrid branch line coupler 101 and L1 WO 88/01984 8 PCT/AU87/00296 comprises a transmission line circuit arranged in a general rectangular configuration. Arranged at one end of the branchline coupler 101 is a first output port 104 and arranged at the other end of the branchline coupler 101 is a second output port 105. The outlet port 104 is connected to a termination 106. The- output signal received at output port 104 is 900 out of phase with that which is received at output port 105. However, it should be noted that in some embodiments of the invention, a single output port could merely be coupled to the transmission line 90 without the provision of two output signals which vary in "phase by 90 e The reference arm 18 and sample arm 16 are separated by a septum 99 to prevent electromagnetic interference between the two arms.

The sample arm 16 of the spectrometer comprises attenuator 20 which is identical to the attenuator 38 previously described and therefore will not be further detailed herein save to say that the reference numerals used in the attenuator 20 corresponds to those'utilized in the attenuatcr 38. Upon receipt of a suitable current at input port 88-of attenuator 20, the signal at terminal 3 of circulator 81 is received on transmission line 110.

Directional coupler 50 is arranged in proximity to transmission line 110 and is identical to the d1irectional coupler 50 arranged in the reference arm 18. The purpose of the directional coupler 50 in the sample arm 16 is the same as that in the reference arm 18. The microwave signal on line 110 is received by circulator 130 which has microwave transmission line 121 connected to its second terminal via DC decoupling capacitor 122. The other end of the line 121 is connected to port 123 which in turn is coupled to loop gap resonator 22 (Figvre 1) so that the radiation can be received by the sample. The radiation is affected by the sample under conditions of paramagnetic resonance and then received by the circulator 30 which directs the radiation from its third terminal to an in phase divider 124 defined by a microwave WO 88/01984 9 PCT/AU87/00296 transmission line which splits the signal and supplies thle signal to two ports 125 and 126 having the same phase, The in-phase divider 124 is a second Wilkinsc-, divider.

Figure 4 shows a cross-sectional view of part of the spectrometer of Figure 2. The part of the spectrometer shown in Figure-2 is formed on an aluminium base 130. The base 130 is provided with recesses 132 (only one of which is shown) for receiving one of the magnets 134 of the circulators, such as the circulator 30. Attached to the aluminium base 130 is a microwave substrate 136 which is provided with a copper layer on both of its sides. The board 136 may be coupled to the base 130 by screws, conducting adhesive or any other suitable devices. The top copper layer of the board 136 is etched to provide the lines 84, 91, 110, 121 and 124 shown in Figure 2.

Those lines are then coupled to the appropriate terminals, of the circulator 30 and the circulator 81 shown in Figure 2 by copper ribbons 138 or by the DC~ decoupling capacitor previously mentioned. in the embodiment shown in Figure 2 ,three b~oards similar to the L~oard 136 are provided. The other -two boards 137 and 139 have the Wilkinson divider 14 and the lines 77, 80, 84, 91 and 101 etched thereupon respectively.

It should be understood that whilst three separate boards are shown in Figure 2, a single board or more than three boards could be utilized.

Figure 2 shows i6 4 electrical form to the left of the dotted line, the structur'_ Which has been disclosed with reference to Figure 2. on Figure 3 the same reference numerals- as used on Figure 2 have been included, Figure 3 also details the detection portion of the embodiment of Figure 2. The signals which are rereived at outputs 104, 105, 125 and 126 are applied to tWo sets of 2 balanced mixers 32 and 141. One of the balanced mixers 140 receives the 90 0 phase shifted signal from the branchline coupler 101 and the signal from the output 125 and the other receives the 0 0 signal from the branohline coupler 101 and the signal from the output 126 from the main or sample arm 16.

The first set of balanced m.4cers discriminates audio frequency WO 88/01984 10- PCT/AU87/00296 encoded signal from the microwave carrier signal. The output from the balanced mixers 32 is then applied to the second set of balanced mixers 141 where low frequency carrier is discriminated from the sample signal. The output of the second set of balanced mixers 141 is applied to analogue to digital converter 142 and then to a calibration/correction unit 143 which is computer controlled to compensate for any variations in signal which may be detected by the monitoring computer system. The output of the calibration/correction unit 143 is then applied to circuits 144 for determining phase differences between the reference and sample signals so that dispersion Rnd absorption properties of the sample can be determined. Squaring circuits 145 and adding circuit 149 also receive the signal from unit 143 so that, an amplitude signal can be determined. A square root circuit 151 ma also be provided to produce a magnitude Value in a more convenient form. Thus the information which is received is a complete description of the E.S.R. properties of the sample. In ,kaddition. it is possible to ,incorpd~rat- signal detecticn as a function of, the phase and frequency of the magnetic field modulation to produce saturation transfer EPR.

Figure shows a flow sheet which generally outlines the computer control of the spectrometer. In Figture 5 a control computer 150 is used to control microwave energy level, magnetic field sweeping, as well as for controlling the Voltages and currents which are applied to the input ports 76, 88 and 85 of the device shown in Figure 2. A data acquisition computer 160 is provided for coupling with the control computer unit and the sample cavity for data acquisition and signal processing. Arithmetical operations are performed in. a digital signal processor unit 170 and the processor 170, data acquisition computer 160 and control computer 150 are coupled with a master computer for displaying informati.on and providing user Input.

At an exa~mple Figure 6 shows some typical EPP, spectra for sapphires., WO 88/01984 1-PC1'/AU87/00296 The invention can include the attachment of a pulse generator to a microwave switch to be inserted between isolator 12 and coupler 14 .Figure 1) which would provide means of carrying out time dynamic ESR followed by use of fast Fourier transform on the local computer to generate the spectrum etc. The compact microwave circuit, in contrast to the bulky waveguide systeris conventionally used at 10 GHz should offer some advantage in terms of reduction in pulse degradation. It would have to be established what effective frequency range was obtained in the FFT of the free induction decay.

In relatlion to Electron Spin Echo Envelope Modulation studies the use of lower frequencies (and magnetic fields) should prove beneficial in interpreting frequency dependent line width information in conventional CW ESP, and, in the case of proteins, provide a more acid test of the role played by protons distant from the paramagnetic centres.

The pulse mode has the benefit in that a fixed magnetic field provided by a permahent magnet is-all that would be needed.

The pulse mode can be implemented with ease owing to.

the simple nature of the active, electronically controlled attonuAtort which would also be employed for the purpose of switching.

The free-standing microwave bridge could be produced at a given frequency, or range of frequencies, for Use as an accessory to any existing commercial or purpose btiilt ESR spectrometer.

Using octave bandwidth design and careful phase compensation, wide band bridge capability should be obtainable.

All systems envisaged are expected to be portable, capable of battery operationt and able to be carried anywhere in the World.

WO 88/01984 12 PCT/AU87/00296 Since modification within the spirit and scope of the invention may readily be effected by persons skilled within the art, it is to be understood that this invention is not limited to the particular embodiment described by way of example hereinabove.

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Claims (8)

1. 2. The spectrometer of claim 1, wherein the spectrometer includes an isolator for receiving radiation from the microwave source to prevent reflected radiation from returning to the source, a divider for directing some of said radiation to the sample and reference arms, an attenuator arranged in the sample arm between the divider and the circulator; a phase shifter and a second attenuator arranged in the reference arm between the divider and the detector.
2. 3. g A spectrometer according to Claim 2, wherein said sample arm and said reference arm formed as an integrated microwave bridge comprises; said divider having a first output portion coupled to said attenuator, said attenuator being coupled to said circulator by a microwave transmission line, said circulator being coupled to at least one output by a second microwave transmission line; said divider having a second output portion coupled to said phase shifter, said phase shifter including a second circulator and said second attenuator including a third circulator, said second circulator being coupled to said third circulator by a third microwave transmission line, a fourth microwave transmission line coupling an output of said third circu.ator to at least one second output.
3. 4. The spectrometer of Claim 3, wherein said sample arm includes a directional coupler, said directional coupler comprising said first microwave transmission line and a fifth microwave transmission line which has an output terminal at L_ i WO 88/01984 14 PCT/AU87/ 00296 one. end and a termination at the other end, a portion of said fifth 11-ransmission line being arranged in proximity to said f'irst transmission line such that a signal can be induced in the fifth microwave transmission line. A spectrometer according to Claim 3, wherein said reference arm includes a directional coupler, said directijonal Coupler including a portion of said fourth microwave transmission line and a sixth microwave transmission line, at least a portion of said sixth microwave transmission line being arranged in proximity to said fourth microwave transmission line so that a signal can be induced in said sixth microwave, transmission line, one end of said sixth microwave transmission line being coupled to an output and the other end being coupled to a termination.
4. 6. The spectrometer of Claim 3, wherein said divider comprises a microwave transmission line having said two output portions.
5. 7. The spectrometer accor~ding to anyone of Claims 3 to 6, wherein said microwave' transmission lines are etched from a conductive substrate.
6. 8. The spectzometer of Claim 3, wher~in said at least one output conprise5 a pair of outputs which receive a pair of signals of the same phas-:,e and said at least one second output comprises a second pair of signals which receive outputs having a phase difference of 900
7. 9. The spectrometer of Claim 8, where one of said first pair of outputs and one of said second pair of outputs are coupled to a first balanced mixer and the other of said first pair of outputs and the other of said second pair of outputs are cQuapled to a second balanced mixer, said balanced mixers being connected to a second pair of balanced mixers and said second pair of balanced mixers being connected to said detector. WO 88/01984 PCT/AU87/00296 The spectrometer of Claim 9, wherein said detector comprises means for determining both the phase and magnitude of the output signals received at said outputs to enable both absorption and dispersion characteristics of the sample to be determined.
8. 11. The spectrometer of Claim 1, wherei-n the detector comprises two pair of balances mixers, an analogue to digital converter coupled to said mixers, a calibration and correction unit coupled to said converter, a pair of squaring circuits coupled to said unit, an adding circuit coupled to said squaring circuits to enable absorption characteristics of the sample to be determined and phase difference determining circuits coupled to said unit to enable dispersion characteristics of the sample to be determined.