EPR spins its electrons

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Review

EPR spins its electrons Electron paramagnetic resonance (EPR) seems atfirstglance to be very limited in scope because of its requirement that a species have at least one unpaired electron. A surprisingly large number of species, however, have unpaired electrons, including organic and inorganic radicals, many transition metal complexes, and defect centers in solids. EPR has been used for applications such as studying transition metal complexes, spin labeling, spin trapping radiation dosimetry archaeological and forensic dating and oximetry (oxygen concentration measurements) In brief, EPR involves placing a paramagnetic species in an external magnetic field, which causes a splitting of the electron magnetic spin states. A second magnetic field at microwave frequencies is then applied to the sample. In the usual experiment, the microwave frequency remains constant, and the external field is slowly swept through resonance. When the energy of the microwave magnetic field equals the energy separation created by the external magnetic field, EPR is observed absorption or emission of Gil"

i.d. quartz tubes, although aqueWidely applicable bbu toous9-mm solutions require use of capillaries or special flat cells because water absorbs miesoteric technique crowave energy. Analytical Chemistry asked Gareth R. keeps field of Eaton, professor of chemistry at the University of Denver, and Michael K. Bowman, staff scientist at Pacific Northwest manufacturers National Laboratory, for their advice for EPR purchasers. small When asked how the market has ergy as the spin state populations change. To ease detection, the external field is often modulated, most commonly at 100 kHz, and a phase-sensitive detection scheme is used, which results in the characteristic derivative line shapes. Because the resonant frequency depends on the magnitude of the external magnetic field, frequencies ranging from the radio to the infrared can be used. However, the most convenient frequency has proven to be around 9-10 GHz, in the microwave region of the spectrum known as the X-band, because it combines reasonable sensitivity with easy-to-handle sample sizes. Samples are usually prepared in 3-

changed in recent years, Bowman said, "In the lastfiveyears, it's really been the international market that's changed. Until recently, many of the applications of EPR could be met with the old standard instruments made by Varian or Bruker many years ago. In fact, there was a macho thing about keeping your spectrometer limping along. But with the advent of pulse techniques, things have gone beyond what you could do with the older instruments. Because of a lack of funding, U.S. scientists haven't been able to keep up with their counterparts in Europe and Japan Hopefully things will pick UD' otherwise Americans will be left using old technique's on old instruments"

Analytical Chemistry News & Features, May 1, 1996 323 A

Product

Review

Table 1. Summary of representative products Table 1 . Summary o

.

l a

Model Company

EMS 104 EPR Analyzer Bruker Instruments Manning Park 19 Fortune Dr. Billerica, MA 01821-3991 508-667-9580 www.bruker.com

Model 8400 Micro-Now Instruments 8260 N. Elmwood St. P.O. Box 1488 Skokie, IL 60076 708-677-4700

Model 8300A Micro-Now Instruments 8260 N. Elmwood St. P.O. Box 1488 Skokie, IL 60076 708-677-4700

Price Sensitivity With TE102(Q = 6000) With high-Q resonator Microwave bridge Oscillator type Frequency Frequency stability Power output Elec. tuning range Standard resonator Electromagnet Maximum field Pole gap Field stability

$66,000

$60,000-$65,000

$60,000-$65,000

EMX Series Bruker Instruments Manning Park 19 Fortune Dr. Biilerica, MA 01821-3991 508-667-9580 www.bruker.com $121,000

2 x 10 10 spins/0.1 mT Not available

5 x 101° spins/0.1 mT Not available

5 x 1010 spins/0.1 mT Not available

7.3 x 10 9 spins/0.1 mT 5.0 x 109 spins/0.1 mT

Gunn diode 9.7-9.9 GHz INA 25 |iW-25 mW INA TM110 cylindrical

Gunn diode 9.1-9.9 GHz INA 150 mW max. JNA TE102 rectangular

YIG-tuned solid state 8.8-9.6 GHz INA 150 mW max. JNA TE102 rectangular

Gunn diode 9.2-9.9 GHz 1x10" 7 @OdB 400 mW max. 60 MHz TE102 rectangular

0.348 T 15 mm 1 x 1 0 - 4 G/K

0.7 T 12.7 mm ±10ppm

0.6 T 45 mm ±50 mG

Sweep time

0.67 s-90 min

10s-60min

0.5, 1, 2, 4, 8, 16 min

0.64 T (6-in. pole diameter) 60 mm Short term, 1 x 10-6; long term, 2 x 1 0 - 6 / ° C 0.16 s-21,500 s

Sweep width Scan linearity

0-20 mT 0.1% of scan range Single-phase power outlet (220V/2Aor110V/4A) Ajr 50 kHz

0-0.6 T ±0.5% of scan range less than 1 kG 1.5 kW

0.01 G-max. field 0.01% of sweep range

Power supply

0-0.6 T ±0.5% of scan range less than 1 kG 0.5 kW Air to 0.4 T or water to 0.7 T 100 kHz

Water 100 kHz; other modulation frequencies available

Optional 6 kHz-100 kHz in 100 Hz steps; 1st/2nd harmonic; optional 100 Hz-6 kHz module

Software package provides full computer control of all instrument parameters and datamanipulation routines, scaling/ smoothing, addition/subtraction, integration, and plot and store; requires 486 or higher PC-compatible computer Variable temperature controllers

Keypad allows use without computer but may be interfaced with 486 or higher computers; Scientific Solutions software package provides full computer control of all instrument parameters and data manipulation Variable temperature controllers; NMR Gaussmeter

PC-compatible; Pentium: WINEPR and WIN-ACQ software package with automated bridge and cavity tuning, data acquisition, automation routines, datamanipulation routines, including curve fitting, baseline correction, and integration Seven standard magnets with pole gaps 56 to 102 mm; 10 other X-band microwave bridges; L-, S-, K-, and Q-band bridges; field frequency lock; NMR Gaussmeter: temperature controller; SimFonia spectral simulation program

Parallel processors (transputers) in each module connected by high-speed bus provide multitasking operations without active PC participation 404

Cooling Signal channel

Data system

Integrated keyboard operation; can be linked to any AT-compatible PC with RS232 interface; optional WIN-EPR software

Options

Magnet temperature control system

Special features

All results time and date stamped

Small size makes instrument portable; can be used for field measurements

Components can be sold separately to update existing systems

Reader service number

401

402

403

I N A = Information not available at press time

324 A

Analytical Chemistry News & Features, May 1, 1996

1 kW

The players

JES-RE1X JEOL USA P.O. Box 6043 11 Dearborn Rd. Peabody, MA 01961-6043 508-535-5900 www.ieol.com $87,000

JES-TE300 JEOL USA P.O. Box 6043 11 Dearborn Rd. Peabody, MA 01961-6043 508-535-5900 www.jeol.com $164,000

ESP 300 E Bruker Instruments Manning Park 19 Fortune Dr. Billerica, MA 01821-3991 508-667-9580 www.bruker.com INA

Not available I x 101° spins/0.1 mT

Not available 7 x 109 spins/0.1 mT

7.3 x 109 spins/0.1 mT 5.0 x 109 spins/0.1 mT

Gunn diode 8.8-9.6 GHz 1x10-6 0.1 uW-200 mW INA TEpn cylindrical

Gunn diode 8.8-9.6 GHz 1x10-6 0.1 nW-200 mW INA TE011 cylindrical

Gunn diode 9.2-9.9 GHz 1x10"7@OdB 400 mW max. 60 MHz TE102 rectangular

0.65 T 60 mm Short term, 0.3 uT; long term, 1 x 10-5/°C h 100 ms-20 s internal

1.4 T 75 mm Short term, 0.3 uT; long term, 1 x 10-5/°C h 0.1-20 s

1.5 T(10-in. pole diameter) 72 mm Short term, 1 x 10" 6 ; long term, 2 x 1K~6/°C 4 ms-21,500 s

±10 mT to ±50 mT INA

±0.01-±500 mT INA

0.016-max. field 0.01% scan range

3-phase 200 V, 50/60 Hz, 2 kVA

3-phase 200 V, 50/60 Hz, 20kVA Water 25,50,100 kHz; optional 80 Hz; optional 2nd harmonic

12 kW

Water 25, 50, 100 kHz; optional 2nd harmonic

Workstation with UNIX operating system, 660-MB hard drive, tape drive, HP plotter, and ESR dataapplications program available separately

Variable temperature controllers, I I other X-band resonators, as well as Q-band, L-band, and ENDOR resonators, UVradiation apparatus

High-sensitivity cavity, simple key operation, special Gunn diode exclusively used for EPR

405

Water 1.56, 3.12, 6.25, 12.5, 25, 50, 100 kHz; 1st/2nd harmonic; optional 37, 375, 1 kHz, 6 kHz module Workstation with UNIX operatESP 3240 Data System: ing system, 660-MB hard drive, 68040 CPU, OS-9 operating tape drive, HP plotter, and ESR system with automated bridge data-applications program avail- and cavity tuning, data acquisiable separately tion routines, and complete set of data-manipulation routines; software structured to support graphic system in real time Field modulation at 80 Hz; pole Seven standard magnet sizes gaps of 117, 60, or 50 mm; vari- with pole gaps from 56 mm to 102 mm; 10 other X-band able temperature controllers; attachment kits for timebridges; bridges for L-, S-, K-, resolved and pulsed ESR; 11 Q-, and W-bands: CW, stoother X-band resonators as well chastic, and pulse ENDOR: as Q-band, L-band, and time-resolved, pulse, and FTENDOR resonators; magnet EPR at X- and W-band; field rotation bases frequency lock; NMR Gaussmeter; variable temperature controller; SimFonia spectral simulation program; WINEPR data-processing software Built-in frequency counter; 2 MHz digitizer for kinetics touch-screen operation; highwork; rapid field scan capability; can be upgraded with all sensitivity cavity available accessories and options 407 406

When Michael Bowman purchased an EPR spectrometer because it no longer "made economic sense to build something that someone else had spent millions to develop," he discovered that "there are not many companies out there making or at least selling a research-grade instrument. We had a really tough time getting a competitive bid." Because the market is relatively small, only three companies Bruker, JEOL, and Micro-Now Instruments manufacture X-band EPR instruments. Bruker has the largest market share and according to Eaton is the undeniable leader in the field; JEOL and Micro-Now continue tofightfor market share Micro-Now has focused on providing lower-priced portable models for service labs and academic researchers interested in only one type of measurement. According to Clarence Arnow of Micro-Now, the company believes that there is a market for the lower-priced models, and it provides lightweight, compact systems that can perform a variety of measurements. Eaton says he has used the Micro-Now 8400 for undergraduate instruction and W3.s very pleased with its performance. JEOL believes it has found its niche in the reliability of its overall system, to which Robert A. DiPasquale, national sales manager for JEOL's analytical instrument division, credits the electronics in their spectrometers. Like Micro-Now, JEOL tends to sell its instruments to service labs and academic researchers who dedicate the instrument to one type of measurement. Arthur Heiss, vice president for EPR at Bruker Instruments, agrees that the lowerpriced EPR spectrometers have a place in the market, but he feels that Bruker's forte is providing high-reliability research instrumentation for a wide range of applications and users. Bruker has recently introduced a series of lower-priced instruments for routine analysis to supplement its research line of EPR instruments. Table 1, although not intended to be comprehensive, lists representative instruments manufactured by the three companies. EPR components

The primary components of the EPR spectrometer are the microwave bridge, the resonator, and the electromagnet. Because S/N, almost always the dominant concern, is determined by the resonator and the bridge, these two components are especially important.

Analytical Chemistry News & Features, May 1, 1996 325 A

Product

Review

The microwave bridge. The frequencc used most commonly in EPR is 9.5 GHzz Although klystron tubes, which use an electron beamfroma thermionic cathode appro priately modulated to generate a microwavefrequency,were the industry standard for many years, the difficulty in obtaining klystrons has made solid-state oscillators (Gunn diodes) the new standard. "An enor mous amount of engineering has gone into stabilizing the Gunn diode," says Eaton. According to Heiss, Brukerfinallyswitched to Gunn diodes when the company developed a new Gunn oscillator with the wide tuning rcinsre cincl low FIVI noise required to replace the klystron X-band bridges are the most popular because they combine high sensitivity with convenient sample sizes, but bridges are also available for a variety of other frequency ranges. As the frequency increases, the resonance conditions dictate that the sample size decrease, but the sensitivity increases. Low frequencies are appropriate for biological samples such as organs and small animals, whereas higher frequencies are appropriate for highsensitivity analysis of small samples and for providing higher spectral dispersion. The resonant cavity. The resonator, the part of the instrument in which the sample is placed, is often characterized by a quality (Q) factor, a figure of merit relating the maximum energy stored by a cavity to the amount of energy dissipated with each microwave cycle. Energy can be lost to "lossy" samples, such as water. Two types of resonators are commonly used in EPR instruments: the cylindrical TE011 and the rectangular TE102. The TE011 resonator has a significantly higher (roughly three times higher) Q than the TE102 2an is better for low loss samples. However, the TE102 remains the industry standard because of its suitability for many types of experiments, adaptability to a wide variety of samples and use in many theoretical calculations because of its well-defined geometry. In addition to these two standard resonators, many specialized resonators are also available. Bruker's resonator line includes more than 50 different configurations over the six microwave bands, including such specialized devices as dielectric, split-ring, ODMR, cw (continuous wave) and pulse ENDOR and cylindrical and Fabry-Perot resonators for high frequencies. MicroNow offers standard and specialized resonators in the X-band, including resonators for electron beam sample irradiation. JEOL offers 14 resonators in addition to the TE that it delivers with its instruments. 326 A

The electromagnet The size of the magnet has an impact on the instrument in two ways. First, larger magnets generate a larger homogeneous field volume, which is vital in EPR experiments. Second, the size of the magnet determines the maximum attainable field and, hence, the highest usable microwave frequency. In addition, the size of the pole gap determines the accessories that can be integrated into the system; smaller magnets can preclude the use of some attachments. However, says Eaton, a smaller magnet can be an asset in environments where "real estate" is an issue. They are also more portable than larger magnets, which may even exceed some floor-loading requirements. Finally, when cost is a factor,

tation that provides the necessary nanosecond timescale for pulse programming and digitization. According to their product literature, Bruker and JEOL now offer pulse EPR instruments. However, Michael Bowman said that when he tried to purchase a pulse instrument, "JEOL refused to bid for delivery to the United States." DiPasquale confirmed that, although their marketing literature includes the pulse instrument, JEOL does not sell its pulse EPR instrument in the United States. The pulse techniques have been valuable in the structural characterization of metalloproteins. Electron Spin Echo Envelope Modulation (ESEEM) gives information regarding distant or weakly coupled nuclei. Pulse ENDOR a technique in which the microwaves and rf are pulsed, yields information regarding near or strongly coupled nuclei. The advantages are that, with both techniques, one is freedfromsome of the relaxation time constraints of cw ENDOR and the data nicely complement each other. Bruker has developed the only commercially available pulse ENDOR spectrometers and has been delivering them for the past two Ralph Weber, Bruker's U.S. application scientist, says that "Bruker's development efforts in pulse EPR have focused on making sophisticated 2-D experiments (many of which are impossible on most the smaller, less-expensive magnets make home-built spectrometers) commercially available, both at X-band and now at high attractive alternatives. For maximum frequency as well. Pulse positions and flexibility and resolution, however, Eaton widths can be set with 2-ns resolution on recommends using larger magnets. an 8-ns raster. Additionally, the excitation band widths currently realized on comAdvanced EPR methods mercial and home-built instruments do not Perhaps the greatest recent advances in usually warrant higher resolution." EPR have been in the more specialized techniques of electron-nuclear double resEaton, who has developed a home-built onance, time-resolved EPR, pulse or FT1-ns instrument, admits that there may not EPR and high-frequency EPR. be widespread demand for such resoluThe coupling of each electron spin with tion. "The manufacturers quite properly say the entire world doesn't want that, that the surrounding nuclear spins compliI'm a lone voice out in Denver. From an cates the EPR spectrum. Each nuclear industrial standpoint, they're probably spin, however, is coupled to only one electron spin. By monitoring the EPR sig- right." nal at a specific transition while sweepENDOR, continuous wave, and pulsed ing through the NMR frequencies, a much EPR can be run at high frequencies, which simpler spectrum is observed because provide higher spectral resolution than Xeach additional nuclear spin adds only two band EPR. Ray C. Perkins, Jr.. president peaks to the spectrum. This technique, of Resonance Technologies, a small comknown as ENDOR, has much higher reso- pany offering high-frequency EPR, belution than ordinary EPR spectroscopy. lieves that "VHF will be the technology that makes EPR a more viable industrial Pulse techniques were commercially technique." He says that despite the reavailable for NMR long before they were quired 3.5- to 9.5-T magnets, the space recommercially available for EPR The spin quirements for a system are about the relaxation times are so rapid for EPR that it has been difficult to engineer instrumen- same as an X-band research grade instru-

"... not many companies are eut there making or at least selllng a research-grade instrument."

Analytical Chemistry News & Features, May 1, 1996

merit. "If we follow anything like the history of NMR, new applications [for EPR] will open up. I definitely see routine analytical applications on EPR spectrometers." Applications Because EPR crosses so many interdisciplinary boundaries, it has the unusual distinction of having no "typical" applications. The applications are growing because "unpaired electrons show up in the strangest places," Heiss says. "Any place an unpaired electron is floating around" is a potential application for EPR. Among the applications of interest to chemistry, biology, and medicine are spin trapping, spin labeling, and oximetry. Spin trapping is used to identify radical intermediates in organic and inorganic reactions, most often in biological systems. Spin labeling involves attaching a stable free radical to a macromolecule. Sitespecific spin labeling, in which a cysteine is introduced to a protein as a replacement for essentially any amino acid and is then spin labeled, is a growing application of EPR spectroscopy. Of interest to physicists is the 11 S f

of EPR to study defects in solids and charge transfer in semiconductors well as

the elucidation of microdoma,ins in high-temperature superconductors Which one? 'Try to understand the application and the sophistication of the user," says Eaton. "Do you want something that will be immediately usable by a novice, or do you want something that can grow?" Bowman tells purchasers to "put down on paper what they are going to be doing 80% of the time on that particular instrument. Make sure you have the reliability to do the measurements that you're going to do day-in, day-out. There's a tendency to add on every gadget or gizmo available, but, to get an added capability, you make a compromise somewhere else. What you can do is make [the instrument] harder to operate for the 80 to 90% of what you will be doing on it" If the instrument is intended for a focused application, then optimizing the instrument for meeting that application is the most important consideration. If, however, the instrument is to be used in an open-ended research environment, examine the options for present and future experimentation. "If you're really looking to do as many things as commercial hardware allows, Bruker is the way to go," says Eaton.

The future Eaton says, "If you accept the generalization that during the first 50 years EPR has been a cw technique at room temperature under linear conditions, almost all X-band, then the future is in multifrequency, multiresonance, multidimensional, nonlinear, timedomain, and multiquantum spectroscopy." Perkins sees the future of EPR in the high-frequency regime. "VHF accomplishes the same fundamental benefits for EMR that movement to higher fields have and do accomplish for NMR superior reso~ lution and sensitivity. Many areas of re~ search free radical spin trapping spinlabeline become qualitatively different spec troscopies due to improvements in resolution Metal research is quite promising particularly for those metals that cannot be detected at X-band For these reasons we anticipate that increasing numbers of EMR researchers will augment their efforts with VHF technologies and manv will shift to VHF altogether "

"The future is in multifrequency, multiresonance, multidimensional, nonlinear, time-domain, and multiquantum spectroscopy.y Bowman believes that the EPR market is "going to keep expanding and actually become a lot more differentiated. In the past there have basically been instruments in two bands, X and Q bands." New areas have been opened by research at low and high frequencies. "As commercial manufacturers start to come out with instruments in those niches, people will start buying them," Bowman says. "It's just like the optical spectroscopy market was 20 years ag 0 yOU could buy the old Cary 14 and do just about anything you wanted Now it's common to have a variety of specialized instruments that can do things the old workhorses can't I think the EPR market ing to head in that direction ,oo " Celia Henry

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Analytical Chemistry News & Features, May 1, 1996 327 A