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By dealing with wider issues than is generally found in review papers, this book will provide analytical chemists with insights that are not available by searching the literature for papers on a specific topic. Read more Read less. About the Author Edited by Professor J. No customer reviews. Share your thoughts with other customers. Write a customer review. The following points need to be considered carefully regarding the GC-MS interface.

However, in modern GC—MS systems, various other types can be used that allow identification of molecular ion. For example, an orthogonal TOF mass spectrometry coupled with GC is used for confirmation of purity and identity of the components by measuring exact mass and calculating elemental composition. Nowadays, a GC-MS is integrated with various on-line MS databases for several reference compounds with search capabilities that could be useful for spectra match for the identification of separated components.

While HPLC is one of the most powerful separation techniques available today, the IR or FTIR is a useful spectroscopic technique for the identification of organic compounds, because in the mid-IR region the structures of organic compounds have many absorption bands that are characteristic of particular functionalities, e. However, combination of HPLC and IR is difficult and the progress in this hyphenated technique is extremely slow because the hyphenated technique's absorption bands of the mobile phase solvent are so huge in the mid-IR region that they often obscure the small signal generated by the sample components.

In addition, as a detection technique, IR is much less sensitive compared to various other detection techniques, e. One is a flow-cell approach and the other is a solvent-elimination approach.

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In this case, absorption of the mobile phase induces the interference of the detection of sample component absorption bands, but some transparent region of the mid-IR range produces detection possibility. For example, if one uses a mobile phase of a deuterated solvent such as heavy water or perdeuterated methanol, IR can monitor many organic compounds that have C—H structures in the molecules. The solvent-elimination approach is the preferred option in most of the LC-IR operations.

After the mobile phase solvent is eliminated, IR detection is carried out in some medium that has a transparency for IR light. Generally, KBr or KCl salts are used for the collection of sample components in the eluent, and heating up the medium before IR detection eliminates the volatile mobile phase solvents. There are two types of interfaces for the solvent-elimination approach: diffuse-reflectance infrared Fourier transform DRIFT approach and buffer-memory technique. The separated sample emerging from the column can be identified on the basis of its mass spectral data.

A switching valve can help make a working combination of the two techniques.

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A typical automated LC-MS system consists of double three-way diverter in-line with an autosampler, an LC system, and the mass spectrometer. The diverter generally operates as an automatic switching valve to divert undesired portions of the eluate from the LC system to waste before the sample enters the MS.

MS is one of the most sensitive and highly selective methods of molecular analysis, and provides information on the molecular weight as well as the fragmentation pattern of the analyte molecule.

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The information obtained from MS is invaluable for confirming the identities of the analyte molecules. This qualitative analysis makes it possible to reconstruct an unknown compound from MS data. The ionization techniques used in LC-MS are generally soft ionization techniques that mainly display the molecular ion species with only a few fragment ions. Hence, the information obtained from a single LC-MS run, on the structure of the compound, is rather poor.

Introduction to hyphenated techniques and their applications in pharmacy

However, this problem has now been tackled by the introduction of tandem mass spectrometry MS-MS , which provides fragments through collision-induced dissociation of the molecular ions produced. Nowadays, various types of LC-MS systems incorporating different types of interfaces are available commercially. The interfaces are designed in such a way that they offer adequate nebulization and vaporization of the liquid, ionization of the sample, removal of the excess solvent vapor, and extraction of the ions into the mass analyzer. The two most widely used interfaces, especially in relation to natural product analysis, are electrospray ionization ESI and atmospheric pressure chemical ionization APCI.

With these interfaces, various types of analyzers, e. Each of these analyzers, however, offers varying degree of mass accuracy and resolution. In conjunction with these interfaces, different types of analyzers, e. LC-MS systems do not allow a complete and unambiguous on-line identification of a component, unless it is a well-known natural product, and complementary on-line spectroscopic information is available in databases.

One of the main problems associated with LC-MS is that the quality of response strongly depends on various factors, e. For example, a crude natural product extract generally contains a number of various types of compounds that differ considerably in their physicochemical properties, solubilities, molecular size and stability. It is therefore extremely difficult, if not impossible, to optimize the ionization conditions that can be suitable for all those different types of compounds.


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One way to get around this difficulty is to analyze the extract in different ionization modes. Among the spectroscopic techniques available to date, NMR is probably the least sensitive, and yet it provides the most useful structural Sarker and Nahar information toward the structure elucidation of natural products. However, the use of this hyphenated technique in the analytical laboratories started in the latter part of the s only.

LC-NMR promises to be of great value in the analysis of complex mixtures of all types, particularly the analysis of natural products and drug-related metabolites in biofluids. LC-NMR experiments can be performed in both continuous-flow and stop-flow modes. Magnetic field strengths higher than 9.

The analytical flow cell was initially constructed for continuous-flow NMR acquisition. However, the need for full structural assignment of unknown compounds, especially novel natural products, has led to the application in the stopped-flow mode. In fact, the benefits of the closed-loop separation—identification circuit, together with the prospect of using all presently available 2D and 3D NMR techniques in a fully automated way, have prompted the development of stopped-flow modes, e.

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From this detector, the flow is guided into the LC-NMR interface, which can be equipped with additional loops for the intermediate storage of selected LC peaks. Following passage through the probe-head, the flow is routed to a fraction collector for recovery and further investigation of the various fractions analyzed by NMR.

In most of the LC-NMR operations, reversed-phase columns are used, employing a binary or tertiary solvent mixture with isocratic or gradient elution. The protons of the solvents of the mobile phase cause severe problems for obtaining an adequate NMR spectrum. The receiver of the NMR spectrometer is not quite able to handle the intense solvent signals and the weak substance signals at the same time.

To overcome this problem, solvent signal suppression can be achieved by one of the three major methods: presaturation, soft-pulse multiple irradiation or water suppression enhancement through T1 effects WET presaturation employing a z-gradient. Using eluents that have as few 1H NMR resonances as possible, e. Using at least one deuterated solvent, e. Using buffers that have as few 1H NMR resonances as possible, e.

Using ionpair reagents that have as few 1H NMR resonances as possible, e.


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To date, three main types of data acquisition modes have been introduced: continuous-flow acquisition, stopped-flow acquisition, and time-sliced acquisition. Recent advances in both hardware and software for the direct coupling of LC and NMR have given a new life to this hyphenated technique.

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As a result, this method is a powerful tool used in many areas such as natural products, organic molecules, biomolecules, drug impurities, by-products, reaction mixtures, and drug degradation products. LC-NMR, despite being known for about last two decades, has not quite become a widely accepted technique, mainly because of its lower level of sensitivity and higher cost compared to other available hyphenated techniques. However, the recent advances in technology, especially in relation to the developments in pulse field gradients and solvent suppressions methods, the improvement in probe technology, and the introduction of high-field magnets — MHz have offered new impetus to this technique.

CE is an automated separation technique introduced in the early s. CE analysis is driven by an electric field, performed in narrow tubes, and can result in the rapid separation of many hundreds of different compounds. The versatility and the many ways that CE can be used mean that almost all molecules can be separated using this powerful method.

It separates species by applying voltage across buffer-filled capillaries, and is generally used for separating ions that move at different speeds when voltage is applied, depending on their size and charge. The solutes are seen as peaks as they pass through the detector and the area of each peak is proportional to their concentration, which allows quantitative determinations. Analysis includes purity determination, assays, and trace level determinations. A typical CE-MS system. Separation is achieved through channels etched on the surface of the capillary connected to an external high-voltage power supply that delivers sample to ESIMS.

This technique runs in full automation and offers high degree of sensitivity and selectivity. To obtain a stable ion spray and to avoid electrical problems, the CE power supply is used to produce the potential for the CE separation and the ESI sprayer tip simultaneously. ESIMS detection technique is generally used in most of the CE-MS systems because ESI is considered to be one of the most powerful on-line tools for the analysis of biomolecules, including natural products, providing both the molecular weight and structural characterization of analytes. Rapid identification and characterization of known and new natural products directly from plant and marine sources without the necessity of isolation and purification can be achieved by various modern hyphenated techniques [ Figure 6 ].

Techniques like HPLC coupled to NMR or electrospray ionization tandem mass spectrometry ESI-MS-MS have been proven to be extremely powerful tools in natural product analysis, as they permit the fast screening of crude natural product extracts or fractions for detailed information about metabolic profiles, with a minimum amount of material.

The combined application of various hyphenated techniques even allows the discovery of new natural product, including complete and conclusive structure elucidation, and relative configurations prior to time-consuming and costly isolation and purification process. Some examples of the application of hyphenated techniques in natural products analysis are discussed here. Crude natural product extracts, which represent extremely complex mixtures of numerous compounds, can be analyzed successfully by using appropriate hyphenated techniques.

LC-MS, if the ionization technique is chosen appropriately, can be an extremely powerful and informative tool for screening crude plant extracts. The currently available various types of LC-MS systems allow the analysis of small nonpolar compounds to large polar constituents like oligosaccharides, proteins, and tannins present in natural product extracts. Alkaloids are a large group of nitrogen-containing secondary metabolites of plant, microbial, or animal origin.

Various hyphenated techniques have been used in the analysis of several types of alkaloids to date. With the development and wider availability of bench-top systems, GC-MS has become the method of choice for the analysis of various pyrolizidine and quinolizidine types of alkaloids. Quinolizidine alkaloids, the main class of alkaloids found in the family Leguminosae, have been analyzed by GC-MS recently. However, some hydroxylated pyrolizidine alkaloids need to be analyzed as their trimethylsilyl derivatives. A number of protoberberine metabolites, differing in the number and the placement of various oxygen functions on the aromatic rings, have been identified prior to isolation from the Corydalis cell cultures by LC-NMR and LC-MS.

The coumarins are the largest class of 1-benzopyran derivatives that are found mainly in higher plants. HPLC-PDA can be used successfully in the analysis of various phenolic compounds, including coumarins, because of the presence of significant amounts of chromophores in these molecules.