HPLC-NMR analysis

HPLC-NMR analysis in a closed-circuit reveals the stereochemical information for elucidating the structures of unknown compounds (Albert 2002). In contrast to the technique of off-line separation, sample collection, and peak identification closed-circuit analysis guarantees the absence of isomerization and degradation. Very often only small amounts of sample are available after extraction. 

In addition to H-NMR and 13C-NMR spectra, modem 2-D NMR techniques were employed to assign structures to the isolated dimeric and trimeric lignols. The characterization of compound 20 by a combination of NMR techniques is here shown as an example. All of the 8 possible isomers were found to be present in compound 20. These isomers were separated into two groups by HPLC. NMR analysis of one of... 

Fig. 39. Contour plot of chemical shift vs. retention time and chemigram of the on-line HPLC-NMR analysis of a technical poly(ethylene oxide) (from [210] with permission)...

On-flow HPLC-NMR analysis can also be performed when sufficient material is available. It involves collecting the NMR data continuously as the sample passes through the probe. This is the most efficient method for stmcture evaluation by HPLC-NMR. The NMR data are represented in a 2-D plot where the x direction contains chemical shift information and they direction is representative of the LC retention time. The individual spectra can be extracted from the ID slices along the x axis if so desired. The resolutions in the individual spectra are of somewhat lower quality than in the stop-flow method however, the introduction of the second dimension allows for easy stmcture assignment even for overlapping peaks in the LC separation. As seen in Fig. 19, the on-flow HPLC-NMR characterization shows four distinct sets of resonances. 

An azo coupling reaction of monatomic phenols with diazotized 4-nitroaniline has been investigated. By HPLC, NMR, elemental analysis, UV and IR spectroscopy it has been shown that the azo derivatives of o-guaiacol, o- and m-cresols interact with an excess of diazonium in pH interval of 4,5-9,5 and form corresponding 4,4-di(4-nitrophenylazo)-2,5-cyclohexadien-1 -ones. 

Both MS and NMR coupling to HPLC have been employed for the analysis of p-carotene isomers and determination of lutein and zeaxanthin isomers in spinach, sweet com, and in retina. Capillary high performance hquid chromatography with stop flow connected to NMR (600 MHz) was used for stracture elucidation of all-trans deoxylutein 11 and its isomers.Efforts are in progress to eliminate the remaining major drawbacks such as obligatory use of deuterated solvents in the mobile phase, poor sensitivity, and low throughput of HPLC-NMR analyses. 

DOSY is a technique that may prove successful in the determination of additives in mixtures [279]. Using different field gradients it is possible to distinguish components in a mixture on the basis of their diffusion coefficients. Morris and Johnson [271] have developed diffusion-ordered 2D NMR experiments for the analysis of mixtures. PFG-NMR can thus be used to identify those components in a mixture that have similar (or overlapping) chemical shifts but different diffusional properties. Multivariate curve resolution (MCR) analysis of DOSY data allows generation of pure spectra of the individual components for identification. The pure spin-echo diffusion decays that are obtained for the individual components may be used to determine the diffusion coefficient/distribution [281]. Mixtures of molecules of very similar sizes can readily be analysed by DOSY. Diffusion-ordered spectroscopy [273,282], which does not require prior separation, is a viable competitor for techniques such as HPLC-NMR that are based on chemical separation. 

Thermal degradation of Irganox 1076 in air was studied by means of HPLC-UV/VIS and by preparative HPLC-NMR. At 180 °C cinnamate and dimeric oxidation products are formed, and at 250 °C de-alkylation products are observed [660], On-line LC-NMR hardly covers a real need in polymer/additive analysis, as the off-line option is mostly perfectly adequate for that purpose. 

HPLC-PDA-MS) are already being used. Although HPLC-NMR-MS provides a very powerful approach for compositional and structural analysis, it by no means represents the limit of what is possible in terms of hyphenation. On-line extraction and the attachment of multiple detectors (e.g. IR, F) make the technique even more powerful. Other analytical laboratories such as TG-DTA-DSC-FTIR, TD-CT/Py/GC-MS/FTIR and HPLC-UV/NMR/IR/MS have been put to work, but do not represent practical solutions for routine polymer/additive analysis. 

After removal of the solvent, the residue was eluted through a short silica gel column to remove the catalyst (elution with hexane ethyl acetate = 1 2). The eluent was concentrated in vacuo to give the product 2 (99 % yield) and the diastereoselectivity was determined by HPLC analysis (99 %). The enantios-electivity of the product was determined by lH NMR analysis with chiral shift reagent (+)-Eu(dppm) in CDCI3 and by chiral HPLC analysis (Chir-alcel-OD). 

For solution-phase libraries that are composed of mixtures of compounds, the difficulty of analysis escalates with increasing numbers of compounds. Typically, large mixtures of compounds are not analyzed before screening, whereas small ones may be analyzed for reaction completeness using mass spectrometry, HPLC, NMR, or combinations thereof. The identification and analysis of active compounds from these mixtures is painstakingly tedious, and often complete characterization is possible only after deconvolution procedures and resynthesis of the active compound. For solid-phase libraries, the methods currendy developed are discussed below. 

This is the first attempt to examine family-held ginseng landraces that have been in cultivation for several decades. Clearly, there is evidence of variation between these landraces. Although HPLC-DAD analysis clearly quantified ginsenoside levels and showed variability, it did not present unique characteristics that would rapidly identify each landrace. Currenfiy, Nuclear Magnetic Resonance (NMR) methods are... 

The authors acknowledge L. Ban-Koffi and M. Watson for their technical assistance. The authors thank M. A. Godshall for 6LC and 6PC analysis W.S.C. Tsang for HPLC data A.D. French for x-ray crystal structure analysis L. Kenne for nmr analysis, and B. Lindberg for methylation analysis and helpful advice. 

The coupling of HPLC with NMR represents a powerful method for the high-throughput screening of peptides in mixtures run in stop-flow and continuous-flow modes. It is possible to obtain routine high-quality HPLC/NMR ID NMR data with as little as 5 pg of compound in a chromatogram peak. However, the HPLC/NMR technique cannot be favorably compared to mass spectrometry techniques (HPLC/MS) in terms of sensitivity and speed of analysis. To date, the majority of reports of the use of HPLC/NMR have been for drug metabolites.1 ... 

Vilegas, W. et al., Application of on-line C30 RP-HPLC-NMR for the analysis of flavonoids from leaf extract of Maytenus aquifolium, Phytochem. Anal, 11, 317, 2000. 

By chemical correlation. b By HPLC analysis. c By 1H-NMR analysis. d By X-ray analysis. 

Determined by HPLC and H-NMR analysis. h Determined by isolation (LC) of the pure diastereomers. 

---