Recording cw-EPR spectra
What is the usual way of recording cw-EPR spectra of an unknown sample?
To obtain data that can be properly post-processed and analysed, the signal should not be distorted by either saturation or overmodulation, and it should be as good as possible in terms of its signal-to-noise ratio. Unfortunately, this means that you generally need to optimise parameters for each individual sample, or at least for each individual series of similar samples (in terms of paramagnetic species, solvent, tube dimensions, and temperature). Of course, there are some “informed guesses” for parameters to start from, but don’t fool yourself using these parameters without checking whether you accidentally distort your EPR signal.
Furthermore, if you would like to compare spectra among each other, you will need to ensure that you calibrate your field, usually by recording the spectrum of a sample with known g factor (commonly referred to as “field standard”) or have it properly calibrated (e.g., once by a technician upon setting up the spectrometer in your lab).
Basic familiarity with operating an EPR spectrometer is tacitly assumed. You will find no information how to couple your resonator and similar things, as this strongly depends on the setup you’re using.
Before delving into how to optimise the parameters of a cw-EPR experiment, we need to briefly mention an important but too often overlooked or ignored aspect: recording all relevant metadata during data acquisition. In short: no spectrometer will record all relevant metadata for you automatically, and some will sometimes even record wrong parameters. Hence, make it a habit to always record all relevant metadata during data acquisition in a format that can be directly read by the cwepr package. This is both, a matter of convenience for you when later processing and analysing the data, and a prerequisite for reproducible research and hence integral part of the scientific method.
Note that most commercial EPR spectrometers do not record sufficient metadata for full reproducibility. What is even worse: some tend to write wrong parameter values, as they do not read the parameter values from the devices when the measurement starts/finishes, but only when the operator manually triggers the saving. Hence, you better record crucial metadata by hand in an external file – and understand which values to trust and why.
For further details, see the section on metadata during data acquisition in the user manual.
How to know over which field range to sweep? The answer strongly depends on the kind of sample you’re measuring. Organic radicals usually show rather narrow signals of 5-10 mT centred about a g value of g = 2. Metals are a completely different thing. They have often quite broad spectra and can easily have g values in the range of 1.9–4 and beyond.
In any case, make sure to record enough “baseline” left and right of your actual signal. Beware of your signal having Lorentzian shape. In this case, you will have very broad “wings” with very little intensity, hence need to record quite a bit of “baseline” not to truncate your spectrum. Furthermore, if you plan to correct the phase of your spectra afterwards, having the spectra recorded rather broad is particularly important, as the dispersive component is quite broad as well.
From own experience, people usually tend to record data over a far too narrow field range. For example: if your signal appears to be 5–10 mT wide, as typical for an organic radical, you better record a range of 15–20 mT, assuming a finite Lorentzian contribution to your linewidth.
Microwave power: power sweep
What microwave power to use? Obviously, more is not always better, as you may easily saturate your signals, resulting in broadening that you cannot easily correct for afterwards. Besides that, it strongly depends on your setup what microwave power will yield reasonable results. For organic radicals at ambient temperatures, microwave powers of 1–2 mW will usually be a good first guess.
From a physical point of view, the spin relaxation times determine the microwave power required to saturate your sample. These spin relaxation times or rates are generally temperature-dependent: the lower the temperature, the longer the spin-relaxation time. Furthermore, high-spin systems with S>1/2 generally have shorter spin relaxation times due to spin-spin interactions, resulting in higher microwave power required to saturate your signal. Organic radicals usually have S=1/2 and hence have rather long relaxation times. This means that it is rather simple to saturate the signal of an organic radical, particularly at lower temperatures (i.e. in solid state/frozen solution).
The only way to really know what setting to use to not distort your signal shape is to perform a series of measurements with different microwave power settings. This is colloquially known as “power sweep”.
As 3 dB change in attenuation translates to a factor of 2 in microwave power, 3-dB steps for a power sweep are quite common.
How such power sweep is analysed will be described in more detail later. For now just one important comment:
Saturation sets in much before your signal amplitude decreases again. Often you will not reach the point where you oversaturate your signal so much that its intensity decreases again.
The only valid way of determining whether you are not (yet) saturating is to record two spectra with different microwave power and make sure the intensity difference translates to the same factor as the power difference. Hence, if you record two spectra with 3 dB attenuation difference, your spectra should have an intensity ratio difference of 2, the one recorded with 3 dB less attenuation being twice as large as the other one. Beware that with “intensity”, we mean the area under the curve, i.e. the double integral of the first-derivative spectrum usually obtained in cw-EPR spectroscopy (due to the lock-in detection scheme).
Important aspects to consider when setting up the parameters for a power sweep: As you change the microwave power incident at your sample, your signal size will vary dramatically as well. This will affect other settings, mostly the receiver gain (more on that below), but you will need to take into account that for low power, your signal-to-noise ratio might dramatically reduce as well. Therefore, better try to quickly run a single spectrum at the lowest power setting you have in mind for your power sweep range to see how many accumulations you would need to get a sensible signal-to-noise ratio that allows you to analyse your data.
Modulation amplitude: modulation-amplitude sweep
Next question when recording cw-EPR spectra: What value for the modulation amplitude shall I choose in order not to distort my signal? The seminal answer to this question would be something in line of: Choose the modulation amplitude to be a factor of two (to ten) smaller than your actual line width.
Congratulations. How to know what line width to expect of your signal before you managed to record a distortion-free spectrum? The approach is quite similar to the one for choosing the correct microwave power (see above for details on that). You record a series of spectra with systematically varying the modulation amplitude, colloquially known as “modulation-amplitude sweep”.
The value of choice is the largest modulation amplitude that doesn’t change the signal shape any more. Of course, this is only a valid judgement if your signal-to-noise ratio is high enough to be certain that there really is no effect any more, not that you’re not only not seeing any effect due to too much noise.
The modulation amplitude for a given resonator needs to be calibrated, typically using a standard sample with a very narrow line. Therefore, make sure you’ve loaded and applied the correct calibration file corresponding to the resonator you’re using. Besides that, be aware that due to miscalibration or missing calibration, modulation amplitude settings may not be easily transferred between setups. Technically speaking, they should be comparable if everything was properly calibrated. Reality has it that your setup is not always properly calibrated (and that you sometimes simply forget to check for the correct calibration file to be loaded).
How to automatically analyse such a modulation-amplitude sweep will be detailed later. But if you’re looking for a “quick and dirty” approach, here it is: Record two spectra with different modulation amplitude and plot them scaled to same signal amplitude. If you see no difference in spectral shape, you can be rather sure that you are not overmodulating your signal. If you were already overmodulating, your line width would be a function of the modulation amplitude applied, hence vary with the spectrometer setting.
A word of caution for this type of often automatically performed measurements: The setting of the modulation amplitude will directly and dramatically impact your overall signal strength, with signals being much larger with larger modulation amplitude. Hence, make sure with a short series of scans using the minimum and maximum modulation amplitude setting that you are not clipping your detector (receiver gain setting, see below) and that you still get signals with meaningful signal-to-noise ratio for analysis when recording with the smallest modulation amplitude.
The amplification of the preamplifier in the signal path of your cw-EPR spectrometer can usually be controlled. Typically, the amplification (gain) setting is given in dB values, as the available range spans several orders of magnitude (60-90 dB are a typical range).
Why does the receiver gain setting matter at all? Two reasons: If you set the gain to a value too high, your signal will be amplified by more than what your detector can handle. Hence, you’re clipping your signal and therefore distorting it. If you really overdo this, you will get a flat horizontal line. Much more tricky are those cases where you still overload your detector, but it will respond with a somewhat “smooth” curve that is nevertheless distorted. Usually, one only finds out in retrospect by trying to simulate the data. Sometimes, if you know what you expect, you can judge from the spectral shape that there is some distortion from a too high gain setting.
The opposite end is a receiver gain setting that is too low. The receiver gain, hence signal amplification, has a strong impact on the signal-to-noise figure of your recorded signal. Of course, you’re interested in obtaining the best signal-to-noise ratio, often in the shortest possible time. Therefore, set your receiver gain such that the range the detector covers is not much more than 20 percent larger than your signal. Often spectrometer control software nowadays allows to preview the gain setting.
Signal channel settings
Depending on the type of setup you use, you will usually have to deal with two parameters: conversion time and time constant.
The conversion time is the time the digitizer in the signal channel spends on acquiring signal on every magnetic field point, and is therefore directly connected to the total sweep time for one scan and the number of field points to record. Generally, the longer you set your conversion time, the better your signal-to-noise ratio will become. However, this has some intrinsic limitations. One is the overall stability of your setup that might make it favourable to use shorter conversion times and more individual scans. Another is the lifetime of the paramagnetic species you are interested in. If you are measuring transient species with a limited lifetime, your conversion time should be short compared to the signal change, and if you would like to not only record the signal change on a single magnetic field position, but acquire complete spectra, it should even be much shorter, at least by the factor of field points you record for a single spectrum.
The time constant, on the other hand, acts as a filter to reduce the noise on the acquired signal. Therefore, larger time constants will generally lead to less noisy spectra. However, this filtering comes to a price, and you need to ensure not to accidentally filter your signal and hence distort its line shape. Usually, you will find advice in the literature to set the time constant at least a factor of four smaller than the conversion time, and at least a factor of ten smaller than it takes to pass through the narrowest line of your spectrum. In any case, the value for the time constant should be smaller than that of the conversion time. If in doubt, your best bet will usually be to try it out using a sample with decent signal strength, and make sure the signal shape does not change at all when increasing the time constant, at least not beyond available spectrometer accuracy and repeatability.
Modern spectrometers come equipped with digital signal processing capabilities that are often switched on by default. While generally, there is nothing wrong with digital signal processing, and the EPR community can probably learn a lot from the concepts developed in this (engineering) field decades ago and applied in probably more devices we’re using daily than we can imagine, scientists should usually strive for signals that are as much unprocessed as possible.
Discussing what the term “raw data” actually means would be out of scope of this introduction. But it should be immediately obvious that if we can choose between automatic filtering and no automatic filtering, we should probably opt for the latter, particularly if there is no other way to get the raw, unprocessed data out of the spectrometer software.
Filtering is a very powerful tool, and it has its use in preprocessing of data for complex analyses, such as fitting, finding peaks, etcetera. However, it should never be used to make spectra appear less noisy and hence more pleasant to the eye. This is simply unscientific and should never be done. If there is good reasons to “denoise” your data, clearly state why and what you have done.
Number of field points
How many field points should I record? The simple answer would be something in line of: Enough points to sample even the highest frequency in your spectrum appropriately. But what does that mean?
In old days, there were pretty distinct settings for the number of field points to record with cw-EPR spectrometers. Due to hardware limitations, you could only record powers of two, usually starting with 512 and ending with 8192 points. This simply corresponded to the available memory of your digitizer. A useful side effect of this is that if you would like to apply a Fourier transform to your data (why you would want to do those strange things is a topic for later), you would anyways better make your data points be a power of two.
Back to topic. A somewhat sensible setting would be to record ten data points per modulation amplitude. This appears to be a standard setting on Bruker cw-EPR spectrometers nowadays, and for good reason. The underlying assumption: Your modulation amplitude should be smaller than the smallest line width in your spectrum, and ten data points will be sufficient to faithfully reproduce a periodic signal with a frequency corresponding in its period to that field range.
Generally, as nowadays memory is no real limitation any more, it is always a good advice to record more points than you would usually need, as thus, you sample your noise frequencies with quite some accuracy, rendering it much easier to discriminate between noise and (sharp) signal afterwards by means of Fourier transform or wavelet analysis.
Recording each scan independently
Usually, you will need to record more than one scan to obtain a sufficient signal-to-noise ratio of your signal. The exception proves the rule.
One problem with recording multiple scans can be that many spectrometers average the scans immediately, not saving the individual scans. This is fine as long as everything goes smoothly. However, having personal experience with an environment where you frequently obtain random noise from unknown sources resulting in narrow spikes in your spectra, we strongly recommend saving each scan individually wherever possible.
Some spectrometers do this per se, with others, such as Bruker spectrometers, you can usually perform “field delay” measurements and set the delay to a very short time. A “field delay” measurement is kind of a kinetic experiment where you repeatedly perform a conventional field sweep experiment and save the results as individual rows of a two-dimensional dataset. In this case, all information regarding other parameters of the setup, such as the microwave frequency, that may change during the measurement, are nevertheless lost.
Of course, having recorded 2D datasets instead of the usual 1D datasets makes it less convenient to look at the data, as you first need to average over the second dimension. However, given a software package like cwepr, this can pretty easily be dealt with.
Sample concentration and number of spins
A last factor that definitely determines the quality of the signals you can record is the number of spins present in your sample. Generally, you may be tempted to think that more is better. However, beware of the fact that at higher concentrations, paramagnetic centres are such densely packed that the spins start to “see” each other and couple via both, dipolar and exchange interactions. This generally leads to distortions of your lines.
As a rule of thumb, a 100 uM solution of TEMPO or similar nitroxide spin probe is perfectly fine, and a TEMPO solution of > 1 mM will be inappropriate due to strong spin-spin interactions visible as strongly broadened signals.
On the other end of the scale, the question often arises how many spins you can detect using a conventional cw-EPR spectrometer. Again, the fair answer would be: it depends on the resonator you use and the overall setup and its sensitivity. Furthermore, keep in mind that sensitivity is usually given as spins per field unit (G or mT). The reason for this is obvious: Sensitivity depends on the absolute signal height, but the height of the signal of a paramagnetic species strongly depends on the spectral width, as the integral and not the amplitude is proportional to the number of spins. Typical figures for commercial EPR spectrometers are in the range of 10^10 to 10^11 spins per Gauss. If you are interested in performing quantitative EPR measurements to obtain the absolute number of spins in your sample, have a look at the section on spin quantification in the data analysis section.
I don’t see a signal: what now?
More often than not, you will not see an EPR signal of your unknown sample on first try. However, this does not necessarily mean that there is no EPR signal. Make sure that your spectrometer works and that you did not accidentally made a mistake or set a parameter to a wrong value. You can easily check that your spectrometer works and that you operate it correctly by measuring a sample that you know will give a signal.
EPR standards are a good choice for checking the spectrometer to work correctly, but if you happen to not have access to such a standard sample, coal (from a burned match) or even coffee powder may do well. A freshly prepared solution of TEMPO or some similar nitroxide spin probe will do fine as well. Just make sure to not dissolve it in water or some other solvent with high relative permittivity (dielectric constant), as this will make your dip disappear. Hint: If you dissolve TEMPO or similar in DMSO, you need to use a thin tube, as with a 4 mm tube, you won’t manage to tune your resonator.
If the spectrometer works and the standard sample you measured does show its known signal, there still might be the chance that the other conditions are just not right. If you have high-spin systems, you will usually need to go to rather low temperatures to see an EPR signal at all. As a rule of thumb, Fe-S clusters frequently found in proteins do show up only below 80 K, typically at about 10–20 K.