LC—nuclear magnetic resonance

Liquid chromatography (LC) has already been described and is an excellent separation technique for compounds that are nonvolatile, thermally unstable and relatively polar in nature. The usual detectors for LC are based on refractive index, conductivity, amperometry, light scattering, UV and fluorescence, all of which have been discussed in Section 3.2. However, sometimes it is desirable to have a more powerful detector attached to an LC instrument and, as such, the following combinations are possible LC-infrared spectrometry, LC-atomic spectrometry, LC-inductively coupled plasma-mass spectrometry, LC-mass spectrometry, LC-UV-mass spectrometry, LC-nuclear magnetic resonance and even LC-nuclear magnetic resonance-mass spectrometry. 

New developments in analysis utilizing GC-MS have included GC-FTIR coupled with high-resolution El and CI-MS to identify drinking water contaminants and GC-IR-MS to examine contaminated water, clay, and soil samples. GC-MS, LC-nuclear magnetic resonance (NMR), and LC-MS have been used to identify contaminants in industrial landfill leacheate. Online systems have also been developed such as the measurement of organic contaminants in water samples. MIMS has allowed the development of commercial in situ devices that are now on the market (see above). Almost all combinations of preconcentration, separation, and mass detection have seen significant advances. 

LC-nuclear magnetic resonance (NMR) One information-rich spectral technique that is more suited to the liquid mobile phase of HPLC than to the vapor phase of GC is NMR. LC-NMR has been implemented, but it has significant limitations. To obtain interpretable spectra of unknowns, concentrations in the measurement cell must be higher than with other detectors. The cell must be smaller than the usual NMR tube, so for any but the very highest concentrations of analytes, FT-NMR acquisition is preferred, with each eluted peak being retained in the measmement cell by a stopped-fiow procedme similar to that employed to increase sensitivity in GC-IR (Section 12.8.2). Expensive deuterated mobile-phase solvents are required for proton NMR, which mandates the use of low mobile-phase volume flow columns narrow-bore or even capillary HPLC. LC-NMR is expensive to implement and only just becoming available from commercial vendors at this time. 

When two compounds with very close, or even super-imposable, retention times and identical UV spectra are found in a chromatogram, it is necessary to use more sophisticated detectors that can yield much more structural information without requiring the isolation of the compounds. LC-mass spectrometry (LC-MS) and LC-nuclear magnetic resonance (LC-NMR) are often used, as it will be presented later. 

The role of advances in chromatographic techniques has been a step point in the development of phytochemistry [67]. Because of the complexity of crude herbal extracts, various online hyphenated techniques have been developed for the analysis of the complex mixtures. These techniques include liquid chromatography (LC), mass spectrometry (MS), LC nuclear magnetic resonance (NMR), and LC-NMR-MS [68]. They facilitate the structure determination of unknown constituents in crude extracts. For example, they are of great applicability in the analysis of flavonoids and other phenolic compounds [69, 70]. 

The most common detectors in HPLC are ultraviolet, fluorescence, electrochemical detector and diffractometer. However, despite all improvements of these techniques it seems necessary to have a more selectivity and sensitivity detector for the purposes of the medical analysis. It should be therefore improvements to couple analytical techniques like infrared IR, MS, nuclear magnetic resonance (NMR), inductively coupled plasma-MS (ICP-MS) or biospecific detectors to the LC-system and many efforts have been made in this field. 

LC-MS/MS Liquid chromatography coupled with tandem mass spectrometry LC-NMR Liquid chromatography coupled with nuclear magnetic resonance TLC Thin-layer chromatography... 

AHLs can be tentatively identified by comparison of the unknown with synthetic AHL standards after Thin Layer Chromatography (TLC) in which the plates are overlaid with agar containing one of the AHL biosensors described above [37,39,44,45]. However, for the unequivocal identification of AHLs the use of more powerful methods such as LC-mass spectrometry, nuclear magnetic resonance and infrared spectroscopy as described below are required. 

Hyphenated analytical techniques such as LC-MS, which combines liquid chromatography and mass spectrometry, are well-developed laboratory tools that are widely used in the pharmaceutical industry. Eor some compounds, mass spectrometry alone is insufficient for complete structural elucidation of unknown compounds nuclear magnetic resonance spectroscopy (NMR) can help elucidate the structure of these compounds (see Chapter 20). Traditionally, NMR experiments are performed on more or less pure samples, in which the signals of a single component dominate. Therefore, the structural analysis of individual components of complex mixtures is normally time-consuming and less cost-effective. The... 

LC-MS (liquid chromatography-mass detector) and LC-NMR (liquid chromatography-nuclear magnetic resonance spectroscopy). 

Below we report methodological studies based upon HPLC, GC/FID, GC-MS, LC-MS, matrix-assisted laser desorption ionisation coupled with time-of-flight mass spectrometry (MALDI-ToF/MS), CE, proton nuclear magnetic resonance ( I INMR), RIA and enzymatic colorimetric techniques. 

The system relies upon preliminary fractionation of the microbial crude extract by dualmode countercurrent chromatography coupled with photodiode array detection (PDA). The ultraviolet-visible (UV-Vis) spectra and liquid chromatography-mass spectrometry (LC-MS) of biologically active peaks are used for identification. Confirmation of compound identity is accomplished by nuclear magnetic resonance (NMR). Use of an integrated system countercurrent chromatography (CCC) separation, PDA detection, and LC-MS rapidly provided profiles and structural information extremely useful for metabolite identification (dereplication, Figure 14.1). 

Use of an integrated system incorporating CCC separation, PDA detector, and LC-MS proved to be a valuable tool in the rapid identification of known compounds from microbial extracts.6 This collection of analytical data has enabled us to make exploratory use of advanced data analysis methods to enhance the identification process. For example, from the UV absorbance maxima and molecular weight for the active compound(s) present in a fraction, a list of potential structural matches from a natural products database (e.g., Berdy Bioactive Natural Products Database, Dictionary of Natural Products by Chapman and Hall, etc.) can be generated. Subsequently, the identity of metabolite(s) was ascertained by acquiring a proton nuclear magnetic resonance ( H-NMR) spectrum. 

Many other analytical techniques can be coupled to mass spectrometers. These so-called hyphenated techniques, like GC-MS and LC-MS, include but are not limited to ICP-MS (inductively coupled argon plasma), SCF-MS (supercritical fluid), NMR-MS (nuclear magnetic resonance) and IR-MS (infrared). 

Abbreviations DCM, dichloromethane DIC, 1,3-diisopropylcarbodiimide DIEA, diiso-propylethylamine DMAP, 4-dimethylaminopyridine DMF, IVJV-dimethylformamide ELSD, evaporative light scattering detection HOBt, hydroxybenzotriazole IR, infrared LC/MS, high-pressure liquid chromatography/mass spectrometry NMM, V-methylmorpho-line NMR, nuclear magnetic resonance PyBop, benzotriazol-l-yloxytripyrrolidino-phosphonium hexafluorophosphate SAR, structure-activity relationship TFP, tetrafluorophenol THF, tetrahydrofuran. 

Reagents were obtained from commercial sources and used as received. V-Boc anthranilic acid 13 was purchased from Advanced Chem-Tech and V-Boc-methyl anthranilic acid 14 was purchased from Aldrich. Proton nuclear magnetic resonance ( H NMR) spectra were run at 500 MHz. LC/MS analysis was performed using a Ci8 Hypersil BDS 3-prn 2.1 x 50-mm column (UV 220 nm) with a mobile phase of 0.1% TFA in CH3CN/H20, gradient from 10% CH3CN to 100% over 5 or 15 min and APcI ionization. 

The coupling of LC (liquid chromatography) with NMR (nuclear magnetic resonance) spectroscopy can be considered now to be a standard analytical technique. Today, even more complex systems, which also include mass spectrometry (MS), are used. The question arises as to how such systems are handled efficiently with an increasing cost and a decreasing availability of skilled personal. LC-NMR and LC-NMR/MS combine the well-established techniques of LC, NMR and MS. For each of those techniques, various automation procedures and software packages are available and used in analytical laboratories. However, due to the necessary interfacing of such techniques, completely new demands occur and additional problems have to overcome. 

LC-NMR. Separations using reverse-phase (RP) liquid chromatography are potentially more powerful because samples can be studied without derivatization. Numerous attempts have been made to separate NOM and while most studies exhibit some degree of separation, to date the complete separation of a NOM sample has not been accomplished. Even only partial separation is possible, and it is worth to hyphenate a separation method with structure information-oriented analytical applications. Liquid chromatography combined with nuclear magnetic resonance and preliminary studies with solid-phase extraction were conducted on NOM isolated from freshwater and soil (Simpson et al., 2004). 

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