Ultraviolet hyphenations

Chromatographic methods are also often used as part of systems that are called hyphenated methods, (see Chapter 15) where the output of the chromatographic section is used as the input for an identification method such as mass spectrometry. These hyphenated methods are also most often referred to by their acronyms, for example, GC-MS—gas chromatography-mass spectrometry and HPLC-MS—high-performance liquid chromatography-mass spectrometry. Note that although ultraviolet-visible (UV-Vis) is hyphenated, it is not a hyphenated method in that it does not consist of two different methods of analysis. Hyphenated methods will be discussed fully in Chapter 15. 

The first exposure to spectroscopy for most scientists is ultraviolet/ visible absorbance. As virtually every HPLC chromatograph employed in the pharmaceutical industry uses UV absorbance as the detection method, it is no wonder that the most popular hyphenated technique is HPLC-DAD. DAD spectrographs have been coupled to all liquid-based chromatographic systems including HPLC (preparative, analytical, and microbore), capillary electrophoresis (CE) and supercritical fluid chromatography (SFC). There have been several successes with TLC plates,18 but it is more common for developed plates to be scraped and the sample analyzed offline. 

This limitation has been largely overcome by linking chromatographic columns directly with ultraviolet, infrared, and mass spectrometers. The resulting hyphenated instruments are powerful tools for identifying the components of complex mixtures (see Section 31A-4). An example of the use of mass spectroscopy combined with gas chromatography for the identification of constituents in blood is given in Feature 31-1. 

Whilst spectroscopy techniques can be used on their own to obtain spectral information about a sample they are also commonly incorporated as a detector as part of another technique, for example the use of an ultraviolet absorbance detector as part of a liquid chromatography system. In recent years there has been much enthusiasm for the research and development of hyphenated techniques, that is the interfacing/linking together of two or more techniques, because of the enhanced additional data that can be generated. 

Another development, also with a history in chemistry, is second-order calibration, where rank annihilation was developed for analyzing data from typically hyphenated instruments. This includes excitation-emission fluorescence spectra of different samples, liquid chromatography with ultraviolet (UV) detection for different samples and gas chromatography with mass spectrometric detection for different samples, giving an array. An illustration is given in Figure 10.2. 

For the purification of compounds, methods including molecular filtration, solid phase extraction (SPE, SPME), solvent extraction, and a variety of basic chromatographic techniques (thin layer, low pressure, ion exchange, size exclusion, etc.), HPLC, and GC (with derivatization of nonvolatile compounds) can be used. Additionally, instrumentation to identify compounds is available, such as the different spectrometric applications, including infrared (IR), mass (MS), ultraviolet and visible (UV-Vis), and NMR spectroscopy. In recent years, the so-called hyphenated techniques (combined chromatographic and spectral methods such as... 

The most common method of detection in HPLC exploits the ultraviolet and visible regions of the electromagnetic spectrum (EM see Figure 5.1) in order to detect the analytes of interest. The detectors employed that utilise these regions of the EM spectrum are the ultraviolet (UV) and diode array detectors. However, other, more specific detectors can be used for specialised applications, such as conductivity and refractive index (not discussed here), and with the introduction of hyphenated techniques, mass spectrometry has become a widely used detector method. 

This is a relatively recent addition to the ionisation techniques used with hyphenated liquid chromatography. In this type of detector, a vaporiser converts the eluent (from the LC) into the gas phase, much like with APCI. The difference with this technique is that instead of the production of electrons from a corona, here we have a discharge lamp producing photons (known as vacuum ultraviolet photons) in a narrow range of ionisation energies. 

Several forms of LC have been used for the separation of organotins. These techniques offer a simple, direct separation, which does not require derivati-zation of the species. The main disadvantages are the lower resolution available with LC and the limitations on detectors that can be coupled to LC for organotin analysis. Ultraviolet detection is not appropriate due to its very poor sensitivity for tin however, fluorimetry has been successfully employed. The recent development of the two hyphenated techniques of LC-MS and LC-ICP-MS has provided two sensitive and selective detectors available for LC speciation. 

Spectroscopic techniques used in essential oil analysis comprise ultraviolet and visible spectrophotometry, infrared spectrophotometry (IR), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR), including the following H-NMR, C-NMR, and site-specific natural isotope fractionation NMR. Combined techniques (hyphenated techniques) employed in essential oil analysis are GC/MS, liquid chromatography/mass spectrometry, gas chromatography/Fourier transform infrared spectrophotometry (GC/FT-IR), GC/FT-IR/MS, GC/atomic emission detector, GC/isotope ratio mass spectrometry, multidimensional GC/MS. 

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