Hyphenated NMR Methods

MS provides the added capability of identifying a molecularweight to an unknown metabolite chromatographic peak, which significantly simplifies the NMR structural analysis. Similarly, solid-phase extraction addresses a common limitation of hyphenated techniques. NMR is an inherently insensitive technique requiring a large sample size ( 500 ng) to observe a simple ID NMR spectrum, which may not be achieved for biological 

Presaturation Traditionally, the solvent signal is irradiated for a period of time with a continuous wave rf field Can be easily set up Useful for eliminating single solvent signal. Extremely sensitive to spectrometer stability and shimming. Not easy to suppress multiple solvent peaks simultaneously. Also, suppresses exchange peaks Suppresses NMR signals from the compound that overlaps with the solvent. 

Excitation The solvent resonances are Involved setup, could be auto- Setup is involved. Several 

The most common HPLC solvents used for LC NMR are acetonitrile and water. Both protonated and deuterated acetonitrile and water are commercially available and routinely used, where cost is a major consideration in minimizing the utility of deuterated solvents in HPLC experiments. Commonly, a low percentage of trifloroacetic acid (TEA) is also added to the HPLC solvent system to improve the LC peak lineshape. In some cases, a methanol water solvent mixture replaces the water-acetonitrile system if the compound of interest does not behave well (low solubility, aggregation, broad peak shape). If an MS system is attached to the LC-NMR to monitor the molecular weight than formic acid is preferred as a modifier. Table 12.9 provides a Kst of commonly used hyphenated techniques and their advantages and disadvantages. 

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Another important application of hyphenated NMR methods is to provide insights into processes that affect the separation. Eor example, online NMR detection of the water chemical shift was used to noninvasively probe intracapillary temperatures in CE separations with subsecond temporal resolution and spatial resolution on the order of 1 mm [111]. Lacey et al. [112] followed up this report with a second NMR study using a novel 2-turn vertical solenoidal coil to measure temperature increases of more than 50 C in a chromatographic frit of the type used in CEC. Insights into the mechanisms underlying cITP have also been investigated utilizing online NMR... 

Hyphenated NMR methods are not considered in the present chapter. The interested reader is referred to Chapter 6 or to one of the numerous reviews that have appeared recently that deal with various hyphenated NMR methods [40,61-63]. 

Jaroszewski JW, Hyphenated NMR methods in natural products research. Part 1 Direct hyphenation, PlantaMed7 63 —700, 2005. 

Wilson ID, Griffiths L, Lindon JD, Nicholson JK. HPLC/NMR and related hyphenated NMR methods. In Gorog S, ed. Identification and Determination of Impurities in Drugs. New York Elsevier, 2000 299-322. 

Four general classes of NMR experiments are routinely used to analyze metabolites (1) ID NMR experiments (2) 2D NMR experiments (3) Solvent suppression methods and (4) Hyphenated NMR experiments. The ID and 2D NMR experiments are commonly used for metabolite structure determination. The various solvent suppression techniques (Gaggelli and Valensin, 1993 Hwang and Shaka, 1995 Smallcombe and Patt, 1995) are crucial for dilute metabolite samples where the solvent peak is the most intense peak in the NMR spectrum. These solvent suppression techniques can be incorporated as needed in both ID and 2D NMR experiments. Since their introduction in the 1990s, hyphenated NMR methods have become common tools in the identification of metabolites. These methods include LC-NMR (Albert, 1995 Spraul et al., 1993, 1994), LC-NMR-MS (Mass Spectrometry) (Shockcor et al., 1996) and LC/SPE (solid phase extraction)/NMR (Alexander et al., 2006 Bieri et al., 2006 Xu et al., 2005 Wilson et al., 2006). 

TABLE 12.9 Advantages and limitations of the most common hyphenated NMR methods. 

Wilson, I.D. Griffiths, L. Lindon, J.C. Nicholson, J.K. HPLC/NMR and related hyphenated NMR methods. Progress Pharma. Biomed. Anal. 2002, 4, 299-322. 

In the case of the low abundance of some compounds, there are difficulties with signal overlap. To overcome these difficulties, there have been developments involving NMR hyphenation with techniques such as HPLC and mass spectrometry. In LC/NMR methods of analysis, NMR is used as the detector following LC separation and this technique is capable of detecting low concentrations in the nanogram range. This technique has been reported for the detection and identification of flavanoids in fruit juices and the characterization of sugars in wine [17]. 

Hyphenated heteronuclear shift correlation methods Many of the experiments described thus far can be used as building blocks to create more sophisticated pulse sequences. The earliest variants of hyphenated NMR experiments, such as HC-RELAY, were heteronuclear detected [56]. More recent hyphenated heteronuclear shift correlation methods are based on proton detection and include... 

As liquid chromatography plays a dominant role in chemical separations, advancements in the field of LC-NMR and the availability of commercial LC-NMR instrumentation in several formats has contributed to the widespread acceptance of hyphenated NMR techniques. The different methods for sampling and data acquisition, as well as selected applications will be discussed in this section. LC-NMR has found a wide range of applications including structure elucidation of natural products, studies of drug metabolism, transformation of environmental contaminants, structure determination of pharmaceutical impurities, and analysis of biofiuids such as urine and blood plasma. Readers interested in an in-depth treatment of this topic are referred to the recent book on this subject [25]. 

Recent trends indicate an increased use of hyphenated NMR techniques, especially those involving NMR in combination with chromatographic methods such as HPLC, GPC, or SEC.P°l Online SEC- and GPC-NMR have found useful applications in the direct determination of molecular weight and molecular weight distribution. ... 

Sampling, sample handling, and storage and sample preparation methods are extensively covered, and modern methods such as accelerated solvent extraction, solid-phase microextraction (SPME), QuEChERS, and microwave techniques are included. Instrumentation, the analysis of liquids and solids, and applications of NMR are discussed in detail. A section on hyphenated NMR techniques is included, along with an expanded section on MRI and advanced imaging. The IR instrumentation section is focused on FTIR instrumentation. Absorption, emission, and reflectance spectroscopy are discussed, as is ETIR microscopy. ATR has been expanded. Near-IR instrumentation and applications are presented, and the topic of chemometrics is introduced. Coverage of Raman spectroscopy includes resonance Raman, surface-enhanced Raman, and Raman microscopy. 

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. 

In this chapter, spectroscopy is an umbrella term for a variety of complementary methods such as tJV/VIS, IR, luminescence, and NMR, with the object of examining mainly polymer additives in solution after extraction but usually before a chromatographic separation. On-line spectroscopic detection hyphenated to chromatography is dealt with in Chapter 7. 

Hyphenation of chromatographic separation techniques (SFC, HPLC, SEC) with NMR spectroscopy as a universal detector is one of the most powerful and time-saving new methods for separation and structural elucidation of unknown compounds and molecular compositions of mixtures [171]. Most of the routinely used NMR flow-cells have detection volumes between 40... 

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