Quantitative NMR by the ERETIC method


We have implemented a variation of the ERETIC method designated "digital ERETIC" (S. Akoka and M. Trierweiler, "Improvement of the ERETIC Method by Digital Synthesis of the Signal and Addition of a Broadband Antenna Inside the NMR Probe," Instrumentation Science & Technology, 30, 21-29 (2002)).  In this version of the experiment, a constant amplitude synthetic signal is added to the experimental spectrum in the form of an exponentially decaying shaped proton pulse routed to the carbon coil of the NMR probe.  The synthetic peak will be at -1 ppm (it can be moved if you have a real signal there, although that will seldom be the case). The real signals can be integrated relative to this synthetic signal.  It can be used just like adding a known quantity of some reference material to the NMR solution, except nothing actually needs to be added to, or later removed from, a pure sample.

You can rpar the parameter set ERETIC and be ready to do the experiment.  As set up, the parameters call for a single scan with no dummy scans.  These conditions were chosen to avoid issues with differential relaxation of different protons in the analyte--the first scan is always quantitative.  Unlike most other experiments we do, it is best not to execute the rga command to scale the receiver gain, because the sample is pulsed repeatedly during that procedure.  If you do an rga, wait a couple of minutes before acquiring the data you intend to use.  If you wish to take mutiple scans in the spectrum, change ns to the desired number of scans and I recommend a d1 of at least 60 seconds in that case. Both the default receiver gain and the size of the synthetic signal are chosen to match a typical multi-milligram sample.  The signal can be scaled up or down in absolute size by changing the power level, pl14, of the shaped pulse. The default pl14 is 37 dB and a change of 6 dB units will change its size by a factor of 2 (increasing the number makes the peak smaller). More generally, the change in intensity can be calculated by dividing the change in dB by 20, and then taking the inverse log of that number (e.g. invlog (6/20) = factor of 2).  Don't use a pl14 setting less than 30 in any case.

The only other change you might need to make would be to the carrier position, o2, of the synthetic signal.  The real signals in the spectrum will have phase corrections that depend on the electrical tuning of the probe, which can vary from solvent to solvent.  These changes do not affect the phase of the synthetic signal, but that signal's phase is very dependent on the precise frequency offset between it and the spectrometer carrier frequency o1.  You can set o2 to the nearest 0.01 Hz, and a 0.01 Hz offset will change the phase of the line by about 1 degree; phase correct the real peaks and then adjust o2 to get the best match between the phases of the synthetic and real signals in response to day to day changes in probe tuning.  That can typically be done by varying o2 by 0.01-0.02 Hz (if needed).

To determine the concentration of your sample, phase the spectrum, baseline correct it with abs, then set the integral of the synthetic peak to 21.8.  If you then look at the integral of a peak in the analyte which should count for 1 proton, its intensity will give the analyte concentration in millimolar.  Multiply that value by the solution volume in ml to obtain the number of micromoles present; multiplying that by the formula weight will give an absolute weight in micrograms.  If pl14 does not have the default value of 37, adjust the assigned integral accordingly--for example, if you lower pl14 from 37 to 43, the 6 dB change will reduce the size of the synthetic signal by half.  In that case, assign its integral to be 10.4. 

ERETIC is available as an automation experiment in ICON-NMR with the user-adjustable parameters of d1, ns, and o2.  It would be advisable to run the first sample manually and note any non-default settings, particularly for o2, and then use the experimentally determined values during the automation run.  You can add any other automation experiments that you wish to run on the samples at the same time.

NMR integrals are subject to a few percent error owing to noise in the spectrum, shimming problems giving rise to nonideal lineshapes, and so forth.  Different NMR tubes may also have slightly different inner diameters which will lead to different active volumes and a resultant systematic error in the calculated concentration.  If no effort is made to control these factors, and you don't do multiple runs in order to get statistics, it is probably reasonable to assign 10% accuracy to the ERETIC-derived value.   But it is quite fast and easy to do--certainly a lot faster than drying the sample down and weighing the solid.

The first place the experiment is implemented is on the Bruker 400 at LSRL.  This system works really well for digital ERETIC because the carbon channel and the lock channel are not on the same coil in the probe on that instrument, and we have a 24 position NMR CASE sample changer to automate the experiment there.  We have tested ERETIC on the 400 in SBC, but in the probe there, the carbon and lock channel share a coil.  In this case the lock is severely disrupted during an ERETIC experiment. We found it is still possible to get an ERETIC spectrum, provided the lock and sweep are turned off on the BSMS control panel prior to acquiring the data.  This instrument does not have automation in any case but it may still be useful to use ERETIC there to spot check single samples.

Dave 9/7/06