## 4 What Are Hyphenated Techniques? Hyphenated techniques are so called because they are developed by coupling a chromatographic technique for the separation of compounds with a mass spectrometry system for mass analysis. Chromatography separates individual compounds in complexes such as plant extracts and the mass spectrometry analyses the masses of the individual compounds. With the aid of a database for searching data on retention time in the chromatographic column, mass data and data on the fragmentation pattern of the compound, the compounds are identified. These techniques are used to generate both qualitative and quantitative data on metabolites. They have wide-ranging applications in pharmaceutics, natural products and drug discovery and in studying responses of plants to various stimuli. When the techniques are represented, the hyphen is placed to separate one application, e.g., chromatography/separation, from the other, e.g., mass spectrometry, as is the case with LC-MS--the hyphen is placed between the LC and the MS. Examples of these hyphenated techniques are liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-nuclear magnetic resonance-mass spectrometry (LC-NMR-MS) and others (Figure 2). The coupling of the techniques is necessary to expand the scope of analysis as well as for better precision in detection, identification and understanding of complex analytes. Liquid chromatography-mass spectrometry is developed by coupling liquid chromatography and mass spectrometry, and sensitivity, efficiency and resolving power can distinguish one LC component from another (Figure 2). However, the later developed ultra-performance liquid chromatography (UPLC) system offers shorter run time. Separation of compounds in the LC is determined by the physico-chemical properties of the stationary and the mobile phases. The stationary phase is the column through which the analyte moves during separation and the mobile phase is the solvent in which the analyte is dissolved. Separated compounds enter into the MS, where their masses are determined through searching the database. When mass information is not sufficient to identify a compound, a tandem MS, or an MS-MS, is useful to generate fragments and the fragmentation data supplement the mass data for accurate identification of the compounds. Further resolution of compounds is obtained by the use of various interfaces like the time of flight (TOF) and the quadrupole. A quadrupole mass analyser consists of four conduction rods arranged in parallel. In the middle of these rods is a space which acts as an ion channel. Separation of ions is based on the stability of their flight trajectories through an oscillating electric field generated with a radio frequency voltage. Only ions with a certain mass-to-charge (m/z) ratio are stable enough to pass through the channel, hence the selection of ions which fall within that particular m/z range. Ions outside this range are filtered out and do not reach the detector. In a triple quadrupole, three sets of the quadrupole are arranged, with the first and the third sets performing the mass filtering function described above. The middle quadrupole performs a collision function. The collision function generates fragments and a fragmentation pattern which, when combined with mass information, helps in compound identification. The TOF interface in mass spectrometry enables the m/z ratio of ions to be detected through determining the time it takes for them to fly in the flight tube. The flight is enabled by ionization prior to the entry of the flight tube. The lighter ions travel faster and reach the detector before the heavier ions. From the detection, the m/z of the ions is determined. Ionization takes place in various forms. The various forms are electron impact ionization, fast atom bombardment, electrospray ionization, atmospheric pressure chemical ionization and matrix-assisted laser desorption ionization. The GC-MS platform works similarly to the LC-MS platform.