Analytical HPLC-NMR

Practical Realization of Analytical HPLC-NMR and Capillary-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. 

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. 

Preparative and analytical HPLC were carried out in an ODS column using gradient elution. The gradient was composed of methanol, water and formic acid. The chemical structures of the new pigments were elucidated by UV-VIS, 2D NMR and LC-MS. MS conditions were capillary 3 kV, cone 30 and 60 V, extractor 7 V, sources block temperature 120°C, desolvation temperature 150°C [257],The chromatographic profile of the SEC fraction containing the new pigments is shown in Fig. 2.116. The chemical structures of the new derivatives identified by various spectroscopic techniques are shown in Fig. 2.117. 

The anthocyanin profile of the flowers of Vanda (Orchidaceae) was investigated with a similar technique. Flowers (2 kg) were extracted with 101 of methanol-acetic acid-water (9 l 10,v/v) at ambient temperature for 24 h. The extract was purified by column chromatography, paper chromatography, TLC and preparative RP-HPLC. Analytical HPLC was carried out in an ODS column (250 X 4.6 mm, i.d.) at 40°C. Gradient conditions were from 40 per cent to 85 per cent B in 30 min (solvent A 1.5 per cent H3P04 in water solvent B 1.5 per cent H3P04, 20 per cent acetic acid and 25 per cent ACN in water). The flow rate was 1 ml/min and analytes were detected at 530 nm. The chemical structures of acylated anthocyanins present in the flowers are compiled in Table 2.90. The relative concentrations of anthocyanins in the flower extracts are listed in Table 2.91. It can be concluded from the results that the complex separation and identification methods (TLC, HPLC, UV-vis and II NMR spectroscopy, FAB-MS) allow the separation, quantitative determination and identification of anthocyanins in orchid flowers [262],... 

Lindon JC, Nicholson JK, Wilson ID. Directly coupled HPLC-NMR and HPLC-NMR-MS in pharmaceutical research and development. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences 748, 233-258, 2000. 

The marine environment clearly holds a tremendous potential for the discovery of lead compounds for development of agents active against infectious diseases and parasites. Within the vast resource of marine flora and fauna are new chemotypes to stem the tide of drug-resistant microbes and insects. Tapping this biological reserve depends on the technology to collect, rapidly recognize, and characterize trace quantities of secondary metabolites. Recent advances in life-support systems and analytical instrumentation, notably with CCUBA, HPLC, NMR, and MS have made this possible. 

High-resolution NMR spectroscopy is demonstrably one of the most important methods of structure elucidation with an extensive range of biochemical and chemical applications. The direct linking of HPLC with NMR spectroscopy has been a remarkable success story as discussed elsewhere in this book and has transformed the technique from a research tool to the stage where routine analytical applications are possible [1,2]. As the number of HPLC-NMR systems have increased in user laboratories, there has been a logical... 

If the retention times of the analytes are known, or there is an efficient method for their detection on-line, such as UV, MS or radioactivity, stop-flow HPLC-NMR becomes a viable option. In the stop-flow technique, all the usual techniques available for high-resolution NMR spectroscopy can be used. In particular, these include valuable techniques for structure determination such as 2-dimensional NMR experiments which provide correlation between NMR resonances based on mutual spin-spin coupling such as the well-known COSY or TOCSY techniques. In practice, it is possible to acquire NMR data on a number of peaks in a chromatogram by using a series of stops during elution without on-column diffusion causing an unacceptable loss of chromatographic resolution. 

Based on chemical shifts and peak multiplicities, the on-flow HPLC-NMR characterisation of the majority of the components in the mixture of 27 tripeptides was achieved and demonstrated that this approach is likely to be an effective method for compound mixtures. The elution positions of all of the alanyl-containing peptides were determined, with the exception of A-M-M-NH2, which may have co-eluted with another peptide or may have been synthesised in a much smaller quantity. The only other tripeptides for which assignments have not been obtained are the MY2-NH2 isomers and two of the three M2Y-NH2 isomers. These eluted towards the end of the gradient run and are not as well resolved under these HPLC conditions. Additionally, with changes in the relative chemical shifts of the solvent signals, the intensities of the non-TV-terminal a-CH protons and the methionyl [3-methylene signals from these peptides may have been reduced by the effects of the solvent suppression irradiation of the water and acetonitrile resonances, respectively. With further optimisation of the elution conditions, it is possible that all 27 analytes could have been resolved and characterised. 

The aliquots of the solution-state chemical synthesis samples were directly injected into a standard HPLC-NMR probe by using a robotic liquid handler. The NMR software was used to automatically find and suppress the intense NMR signals from any non-deuterated solvents used, typically using the WET sequence [5]. Unlike the characterisation of impurities in organic compounds (see the next section) or drug metabolites (see the appropriate chapter in this volume) where the proportions of the analytes can be very different, combinatorial chemistry samples tend to be all of similar quantity and this simplifies the analysis in that it is not usually necessary to worry overly about carry-over of material from sample to sample, nor it is necessary to readjust the NMR spectrometer receiver gain after every sample, thus saving considerable machine time. 

Microbial production of secondary metabolites is also an important source of novel therapeutic agents. However, the physiological and biochemical factors that determine the onset of production of a specific secondary metabolite in a particular species are incompletely understood. Generally, a range of analytical techniques, often elaborate, time-consuming and involving extensive sample pre-treatment, have to be developed in order to monitor the details of the metabolic changes and substrate consumption that accompany secondary metabolite production. In order to provide rapid multi-parametric information about the microbial fermentation process, H HPLC-NMR has been applied to characterise microbial metabolites directly in the broth supernatants from a... 

For the on-line SFE-NMR experiments, the set-up shown in Figure 7.2.17 can be used. A main pump serves an HP supercritical fluid chromatograph (G1205A), with analytical HPLC columns being used as extraction cells. The continuous-flow NMR cell is connected between the column outlet and the back-pressure regulator. 

PCBs can be conveniently determined by most of the common analytical techniques which include GC-ECD, GC-HECD, GC-FID, GC/MS, HPLC, NMR, and enzyme immunoassay. Among these, GC-ECD and GC/MS are by far the most widely used techniques for the determination of PCBs in the environmental samples at a very low level of detection. While the former can detect the PCBs at subnanogram range, the mass selective detector (GC/MS) identifies the components relatively at a higher detection range, 10 to 50 times higher than the ECD detection level. GC/MS, however, is the best confirmatory method to positively confirm the presence of PCBs, especially in heavily contaminated samples. Aqueous and nonaqueous samples must be extracted into a suitable solvent prior to their analysis. 

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