Hyphenated NMR Techniques

It seems like more and more people are interested in exploring the utility of flow-through hyphenated techniques.  Why?  More structural information of course!  I mean, really, why would you spend an afternoon preparing a number of samples and running one analytical technique after another when you could conserve analyte AND time running them all in one go???  Separation AND characterization all in the same experiment……seems like a no brainer!

Here is a partial list of the known NMR-hyphenated techniques:

1)      Liquid Chromatography (LC) –NMR – most common method
2)      Gas Chromatography (GC)–NMR – used for gaseous separations but fraught with sampling difficulties
3)      Size Exclusion Chromatography (SEC)–NMR – primarily used for polymer separations – also Gel Permeation Chromatography (GPC)–NMR
4)      Supercritical Fluid Chromatography (SFC)–NMR – Used to avoid solvent suppression methods also Supercritical Fluid Extraction (SFE)–NMR
5)      Capillary Electrophoresis (CE)–NMR
6)      Cation Exchange Capacity (CEC)–NMR
7)      Capillary Zone Electrophoresis (CZE)–NMR
8)      Capillary Isotachophoresis (cITP)–NMR

Hyphenated (or hybrid) analytical techniques were proposed some time ago but didn’t really catch on due to the poor sensitivity of the NMR experiment and the overwhelmingly large solvent peaks that would swamp out the peaks of interest.  Now that technological advancement has improved some vital parameters (e.g., field strength, sensitivity) and effective solvent suppression protocols have been developed, hyphenated techniques are a reality.  This is particularly useful for mass-limited samples (e.g., environmental quantification, toxicology etc.).

The most common hyphenated NMR technique (by a large margin!) is LC-NMR.  I’ll refer to this as LC throughout this text, but high performance liquid chromatography (HPLC) is the most prominent chromatography used due to its utility in simplifying complex mixtures of dilute samples (e.g., natural products, environmental and toxicological studies).  Generally these combination analytic techniques are run by connecting an LC pump in-line with an NMR spectrometer.  Although the spectrometer can be used as the sole detector, there is usually the normal LC-detector (e.g., UV) connected within the circuit.  With this setup, the analyte can be monitored by NMR in two ways:

1)      Continuous flow – numerous NMR spectra are acquired through the duration of the solvent elution.  This generally requires a reasonably high concentration of the analyte.
2)      Stopped-flow – LC flow is stopped while a particular peak’s NMR spectrum is recorded.  This is better if complex, long run experiments (e.g., COSY, HSQC, NOESY etc.) are required and the sample is of a lower concentration.

The challenges for a flow-based LC-NMR system are: 1) overall system control; 2) detection; 3) timing data collection; 4) managing sample volume; and 5) solvent suppression.

The second challenge is essentially just plumbing.  How long does it take for the sample of interest to flow from the LC-detector to the NMR system?  How long does it take to fill the detector cell in the NMR probe with the desired sample?  These are simple fluid dynamics problems that are exacerbated by: (i) the valving and switching required to deliver the analyte stream to the NMR; and (ii) the diffusion of the analyte as the heterogeneous sample flows through a tube.

The third challenge is more subtle.  For maximum sensitivity in NMR data acquisition, it is desirable to have the sample introduced to the probe at the point of highest concentration.  Detecting this point, and then directing the correct sample volume to the NMR probe at the correct time can be extremely challenging and is dependent upon the control offered by the instrumental setup.

Solvent suppression is also necessary to get good data during data acquisition.  The solvent signal will obscure the sample signal unless reduced.  This challenge is a fairly straightforward process in solvents with a single moiety (e.g., water or chloroform) but more challenging multiple moiety solvents (e.g., toluene or alcohols), mixed solvent systems or solvent gradients.  Modern additions have included solid phase extraction (SPE) into the online network to afford LC-SPE-NMR.  SPE facilitates solvent exchange, which allows for the analyte to be concentrated and/or a deuterated solvent to be introduced in place of the proteo solvent in which the LC extraction was performed.

A more intricate on-line system can be made by directing the LC flow through a series of sample collection devices (e.g., test tubes or storage loops).  This allows separate fractions to be collected and manually (or automatically) introduced into the spectrometer.  This can help simplify some of the plumbing/timing problems discussed above and allows for:

3)      Loop collection – This method accounts for timescale differences that can occur between sample elution/collection time and NMR data acquisition time and is perhaps the best for extremely dilute samples, sensitive and precise extractions (where stopped flow may hinder the separation), and for samples requiring long NMR analysis.

Sooooo……what about the NMReady?  Can this benchtop spectrometer be ‘on-line’ as it were??  In fact, the NMReady was designed with a flow-through magnet with the accommodation of these sort of hybrid techniques in mind!

For further reading please refer to:

1)      Albert, K. “On-Line LC-NMR and Related Techniques” Wiley & Sons: New York, New York. 2002.
2)      Albert, K. J. Chromatogr. A. 1999, 856(1-2), 199]
3)    www.spectroscopynow.com/details/education/sepspec10145education/A-Primer-on-LCNMRMS.html?tzcheck=1 [Viewed Oct 17, 2013]
4)    www.chromatographyonline.com/lcgc/article/articleDetail.jsp?id=473130 [Viewed Oct 17, 2013]