A Novel NMR Tube Design for External Referencing Fazale R. Rana and Jack Blazyk Department of Chemistry and College of Osteopathic Medicine, Ohio University, Athens, OH 45701 Catherine M. Sultany University Research and Instrumentation Center, Ohio University, Athens, OH 45701 The most accurate measurement of the chemical shifts of the NMR signals of a sample is made by comparison to an internal standard in the same solution. When the standard cannot beadded directlv to thesample becauseof reactivitv. for example, the cbekical shift values must be assigned relative to an external standard, such as 85% H3POI for 3'P NMR. External referencing can be performed with expensive and difficult-to-clean coaxial NMR tubes, or alternatively, a sealed capillary containing the reference can be placed in the sample solution. The latter method can lead to a t least two orohlems: (1) motion durine sninnine or offcenter placement of the capillary in the PTMR tubereduces homogeneity and tends t o cause broad lines and sidebands, and (2) sensitivity can suffer if the capillary displaces an appreciable amount of sample from the magnetic field. Modern Fourier transform NMR spectrometers make it possible to assign the chemical shift value of an NMR line by comparing the sample spectrum with a previously acquired spectrum of an external standard. Although this approach is not subject to the disadvantages cited, i t m n lead to significant errors in chemical shift assignments due to differences in medium effects on the shielding in the sample and reference
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ple have been determined, the spectrum of the external reference is measured in the presence of the sample using this apparatus. The reference position is stored and the cap assembly then is replaced with a 10-mm NMR cap. The sample spectrum is measured without the reference peak and compared to the stored spectrum of the external reference. The success of this design is demonstrated in Figure 2, which shows the 3LPspectrum of 85%H3POa (spinning rate of 13 Hz) with a line width at half-height of 4.76 Hz, measured in the presence of an aqueous lipid sample using a Varian VXR-400s NMR spectrometer.
1 -5mmap
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30 mmcap
t l o m m ~ ~ ~ t u b e
P ~ S I pipet ~ Y ~ with sealed end
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HIPO. level
-sample
level
Figure 1. A schematic diagram of the lo-mmNMR tubecapassembly used for external referencing.
We describe here a simole aooroach to external referencing that is inexpensive (requiring only a Pasteur pipet and two NMR tube c a ~ s )easilv . removable from the NMR tube. and reusable. As diagrammed in Figure 1,the chemical shift standard (for examnle. 85% HqPOa) is added to a Pasteur pipet whose tip was'sealed in a flame. The pipet is inserted througha right-fittinp. holedrilled in a 10-mm tubecao. This assembly isksed in place of the cap on a standard i0-mm NMR tube. The external reference is positioned ootimallvin the sample by simply sliding the pip& up or down. A 5-&m NMR tube cap is used to seal the reference material in the Pasteur pipet. Once the acquisition parameters for the sam-
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' Jameson. C. J.: Mason, J. in Multlnuclear NMR; Mason. J., Ed.:
Plenum: New York. 1987: pp 42-44. Jameson, C. J.: Mason. J. in Multinuclear NMR; Mason, J., Ed.; Plenum: New York, 1987: pp 79-80. McFarlane, W.; White, R. F. M. Techniques of High Resolution Nuclear Magnetic Resonance: CRC: Cleveland. 1972.
Figure 2 "P spectrum 01 8590 h,Wl contained In lhe Parfaur p.pet measLrs4 In the presence of an aqJeaJs sample of ipopo~ysaccharde and soa&m dodecyl sulfate.
Volume 68
Number 3 March 1991
269
