Analytical techniques, structure elucidation

Since its discovery in 1946, NMR spectroscopy has rapidly advanced as an interdisciplinary technique that employs the principles of chemistry, physics, engineering, medicine and biology. NMR spectroscopy has become one of the major analytical techniques for elucidation of chemical structure in both the liquid and solid phases. 

NMR, nuclear magnetic resonance, is an analytical technique based on the energy differences of nuclear spin systems in a strong magnetic field. It is a powerful technique for structural elucidation of complex molecules. 

The great strength of mass spectrometry as a technique is that it can provide both the molecular weight of an analyte (the single most discriminating piece of information in structure elucidation) and information concerning the structure of the molecule involved. 

Abstract Protoberberine alkaloids and related compounds represent an important class of molecules and have attracted recent attention for their various pharmacological activities. This chapter deals with the physicochemical properties of several isoquinoline alkaloids (berberine, palmatine and coralyne) and many of their derivatives under various environmental conditions. The interaction of these compounds with polymorphic DNA structures (B-form, Z-form, H -form, protonated form, triple helical form and quadruplex form) and polymorphic RNA structures (A-form, protonated form, triple helical form and quadruplex form) reported by several research groups, employing various analytical techniques such as spectrophotometry, spectrofluorimetry, circular dichro-ism, NMR spectroscopy, viscometry as well as molecular modelling and thermodynamic analysis to elucidate their mode and mechanism of action for structure-activity relationships, are also presented. 

Nowadays, analytical chemistry has a large variety of methods, techniques and apparatus at its disposal and is able to play its instruments with high virtuosity. Therefore, the wide range of performance which analytical chemistry can achieve is extremely varied and extends from simple binary decisions (qualitative analysis) to quantitative analysis at the ultratrace level, from structure elucidation and species identification to studies of the dynamics and the topology of multispecies systems by means of temporally and spatially high-resolving techniques. 

Owing to rapid development in analytical techniques, metabolite identification and structure elucidation have become possible even with trace levels of metabolites generated with in vitro or in vivo mammalian systems. However, the microbial bioreactor is still a valuable system for metabolite structure determination, especially when the metabolite of interest presents at a low level in in vitro or in vivo mammalian systems and the isolation from these matrices is hindered by the interference of other metabolites, the parent drug or endogenous compounds, or the structure determination requires appreciable amounts of samples due to structure complexity. 

Hence, direct mass spectrometry techniques, either using El or ESI, appear to be powerful and innovative analytical tools for elucidating the structure of the main biomarkers present in a wide range of waxes and lipids that may be preserved in archaeological objects and in museum works of art. In most cases, they have nevertheless to be cautiously exploited in combination with other complementary analytical techniques. 

Leucovorin, since it is totally reduced, is polarographically inert in a pH 9 buffered solution.63 After acid treatment, three polarographic waves are generated, corresponding to an anodic oxidation of a tet-rahydro compound and two cathodic reductions of unreduced pteridines presumably at least one of these three is a dihydro species. Polarography is useful as a technique in structural elucidation, but analytical data would be difficult to obtain from an acid-treated solution containing several species, each with its own polarographic behavior. 

Modern spectroscopy plays an important role in pharmaceutical analysis. Historically, spectroscopic techniques such as infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS) were used primarily for characterization of drug substances and structure elucidation of synthetic impurities and degradation products. Because of the limitation in specificity (spectral and chemical interference) and sensitivity, spectroscopy alone has assumed a much less important role than chromatographic techniques in quantitative analytical applications. However, spectroscopy offers the significant advantages of simple sample preparation and expeditious operation. 

Mass spectrometry is an analytical technique to measure molecular masses and to elucidate the structure of molecules by recording the products of their ionization. The mass spectrum is a unique characteristic of a compound. In general it contains information on the molecular mass of an analyte and the masses of its structural fragments. An ion with the heaviest mass in the spectrum is called a molecular ion and represents the molecular mass of the analyte. Because atomic and molecular masses are simple and well-known parameters, a mass spectrum is much easier to understand and interpret than nuclear magnetic resonance (NMR), infrared (IR), ultraviolet (UV), or other types of spectra obtained with various physicochemical methods. Mass spectra are represented in graphic or table format (Fig. 5.1). 

The characterization OF catalyst structures has undergone revolutionary developments in recent years. Powerful novel techniques and instrumentation are now used to analyze catalyst structure before, during, and after use. Many of these advances are responsible for placing the field of catalysis on an improved scientific basis. These developments have resulted in a better understanding of catalytic phenomena, and therefore improvements in commercial catalysts and the discovery of new systems. The application of advanced electronics and computer analysis has optimized many of these analytical tools. These developments are especially evident in spectroscopy, zeolite structure elucidation, and microscopy several other techniques have also been developed. Thus, the difficult goal of unraveling the relationships between the structure and reactivity of catalytic materials is finally within reach. 

Pheromone identification is still difficult because the structure of unique compounds present in small amounts in mixtures of similar molecules has to be elucidated. This topic will be discussed in detail by Ando as well as by others, showing nicely the recent progress in analytical techniques. The following chapter by R. Jurenka deals with insect pheromone biosynthesis with special emphasis on lepidopteran pheromones and also covers genetic aspects. The subsequent chapter by C. Keeling et al. describes the hymenopteran semio-chemicals (bees and ants), describing pheromones and allelochemicals. The hymenoptera add a certain flavor to the scene, because now the complexity of social insects with their many interactions comes into play, as well as the multi-level (multi-trophic) signals used by parasitoids. 

Techniques in isolation and structure elucidation of (volatile) semiochemicals from beetles are the same as in other insects. Problems are mainly due to the often very small amounts of target compounds, embedded in large amounts of non-active substances which form a kind of cosmetic formulation for the biologically active principle. Comprehensive reviews of analytical approaches have been published [18-20]. 

Thus, in order to describe adequately any pure substance, its structure must be elucidated. Before the advent of modern analytical techniques, such as X-ray diffraction analysis, structure elucidation was a much more complicated and time-consuming task than the separation and identification of a compound and occupied the life-work of many eminent chemists. 

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... 

The final section, on analytical chemistry, is a combination of structure-elucidation techniques and instrumental optimizations. Instrumental analysis can be broken into several steps method development, instrumental optimization, data collection, and data analysis. The trend today in analytical instrumentation is computerization. Data collection and analysis are the main reasons for this. The chapters in this section cover all aspects of the process except data collection. Organic structure elucidation is really an extension of data analysis. These packages use spectroscopic data to determine what structural fragments are present and then try to determine... 

---