HPLC/NMR

Capillary gc/ms, hplc, nmr, ir, and uv are all analytical methods used by the terpene chemist with a good Hbrary of reference spectra, capillary gc/ms is probably the most important method used in dealing with the more volatile terpenes used in the davor and fragrance industry (see Flavors and spices). The physical properties of density, refractive index, boiling point, melting point of derivatives, and specific rotation are used less frequendy but are important in defining product specifications. 

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. 

Hyphenation of HPLC with NMR combines the power of sepai ation with a maximum of stiaictural information by NMR. HPLC-NMR has been used in the detection and identification of diaig metabolites in human urine since 1992. The rapid and unambiguous determination of the major metabolites of diaigs without any pretreatment of the investigated fluid represents the main advantage of this approach. Moreover the method is non-destmctive and without the need to use radiolabelled compounds. 

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. 

Dachtler, M. et al.. Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina. Anal. Chem., 73, 667, 2001. 

Sidelmann UG, U Braumann, M Hofmann, M Spraul, JC Lindon, JK Nicholson, SH Hansen (1997) Directly coupled 800 MHz HPLC-NMR spectroscopy of urine and its application to the identification of the major phase II metabolites of tolfenamic acid. Anal Chem 69 607-612. 

Mistry, N., Roberts, A.D., Tranter, G.E., Francis, P., Barylski, I., Ismail, I.M., Nicholson, J.K., and Lindon, J.C., Directly coupled chiral HPLC-NMR and HPLC-CP spectroscopy as complementary methods for structural and enantiomeric isomer identification application to atracurium besylate, Anal. Chem., 71, 2838, 1999. 

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. 

Multiple hyphenation ( hypemation ) provides comprehensive spectroscopic information from a single separation. The first doubly hyphenated HPLC-NMR-MS appeared in 1995 [661], and its value is now accepted meanwhile fully integrated on-line LC-NMR-MS and MSn systems (QMS, QTTMS) are commercially available. On-line LC-NMR-MS coupling is by no means trivial. For example, the sensitivity of NMR is limited, while MS is incompatible with non-volatile buffers. The... 

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. 

Note that in some cases one may follow the time course of covalent E-A formation by equilibrium binding methods (e.g., LC/MS, HPLC, NMR, radioligand incorporation, or spectroscopic methods) rather than by activity measurements. In these cases substrate should also be able to protect the enzyme from inactivation according to Equation (8.7). Likewise a reversible competitive inhibitor should protect the enzyme from covalent modification by a mechanism-based inactivator. In this case the terms. S and Ku in Equation (8.7) would be replaced by [7r] and K respectively, where these terms refer to the concentration and dissociation constant for the reversible inhibitor. 

There are two different ways of carrying out an HPLC-NMR experiment ... 

Our standard molecule is however not ideally suited for certain experiments (e.g. magnetic non-equivalence, NOE, HPLC-NMR coupling). In such cases other simple compounds of the same type, compounds 2-7, will be used ... 

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. 

The instrumental setup for capillary HPLC-NMR coupling is shown in Figure 4.6. The capillary pump is connected via 50 pm capillaries between the capillary HPLC pump, the UV detector, and the NMR flow probe. 

FIGURE 4.6 Instrumental setup for capillary HPLC-NMR coupling. (From Hentschel, P. et al., J. Chromatogr. A, 285, 2006. With permission.)... 

Linking techniques together might seem like a good idea in theory but in practise, there can be as many problems as potential advantages. HPLC-NMR does have undeniable use in the field of bio-fluid NMR and in process control in a production environment but we feel that it has little to offer the organic chemist looking to monitor a reaction. 

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