Major Technique 4: Chromatography

Modern work generally uses three major techniques, chromatography, mass spectrometry and spectroscopy, although there is a wide range of other techniques available. 

A pair of amino acids is separated in a column in which the stationary phase is saturated with water and the carrier solvent is methanol, CH,OH. The more polar the acid, the more strongly it is adsorbed by the stationary phase. The amino acids that were separated in this column are (a) HOOCCHNH2CH,COOH and (b) HOOCCHNH2CH(CH,)2. Which amino acid would you expect to be eluted first Explain your reasoning. Refer to Major Technique 4 on chromatography, which follows these exercises. 

The fatty acids measured by these techniques have all been small monomeric molecules. Lamar and Goerlitz [ 125] studied the acidic materials in highly coloured water and found that most of the nonvolatile material was composed of polymeric hydroxy carboxylic acids, with some aromatic and olefinic unsaturation. Their methods included gas, paper, and column chromatography with infrared spectrophotometry as the major technique used for the actual characterisation of the compounds. 

GC- and LC-MS (Fig. 2), although others have also used other techniques including Fourier transform infrared spectroscopy, thin layer chromatography, high-pressure liquid chromatography, and Raman spectroscopy. The major techniques as judged by current number of publications will be discussed below. 

In argentation thin-layer chromatography, a major technique for lipid separations, application is made of the ability of silver to form complexes with lipids. Silver nitrate is incorporated in either the stationary or mobile phase, and separations are based on the type (such as cis-trans) and extent of unsaturation in the lipids. This type of chromatography has been reviewed by Morris. ... 

NMR spectroscopy is routinely used to characterize new compounds, monitor reaction kinetics, measure magnetic susceptibilities, and quantify chemical yields. NMR spectroscopy is also a major technique used to follow exploratory organometallic reactions since thin layer chromatography of fragile, air-sensitive compounds can either be impossible or too tedious. Thus, a wide range of techniques and equipment have been developed to facilitate the use of NMR spectroscopy with air-sensitive compounds. Some essential air-free techniques for a novice in the field are described below. 

Various experimental techniques have come to the fore in copolymer composition studies. The two major techniques employed are infrared (IR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Controlled pyrolysis linked to gas chromatography (Py-GC) and IR spectroscopy is being employed in a growing number of applications. 

A very important aspect of microstructure is the sequence of monomer units in a polymer. This applies whether the polymer is based on a single monomer which is capable of polymerising in different ways, e.g., head-to-head or head-to-tail polymerisation, or whether it is based on two or more different monomers when many variants of monomer sequence are possible. Sequence distribution has an important bearing on the tacticity and other properties of polymers, as will be discussed later. Three major techniques have been used to study sequence problems in polymers, they are pyrolysis-gas chromatography, nnclear magnetic resonance (NMR) and, more recently, secondary ion mass spectrometry (SIMS). 

The major chromatographic techniques can also be categorised according to tbe nature of the mobile phase used -vapour phase chromatography for when a gas is the mobile phase and liquid chromatography for when a liquid is the mobile phase. 

Preparative chromatography has been used for chiral separations for years, but examples of multi-kg separations (and hence larger ones) were rare until recently. The development of SMB techniques (both hardware and simulation software) has made major breakthroughs in this field. The ability of SMB as a development tool has allowed the pharmaceutical manufacturer to obtain kilo grams quantities of enantiopure drug substances as well benefit from the economics of large-scale production. 

Nevertheless, despite the inherent disadvantages of exclusion chromatography, there are instances where it is the only practical method of choice. The technique is widely used in the separation of macro-molecules of biological origin, e.g. polypeptides, proteins, enzymes, etc. In fact, it is in this area of biotechnology where the major growth in HPLC techniques appears to be taking place. 

Despite its widespread application [31,32], the kinetic resolution has two major drawbacks (i) the maximum theoretical yield is 50% owing to the consumption of only one enantiomer, (ii) the separation of the product and the remaining starting material may be laborious. The separation is usually carried out by chromatography, which is inefficient on a large scale, and several alternative methods have been developed (Figure 6.2). For example, when a cyclic anhydride is the acyl donor in an esterification reaction, the water-soluble monoester monoacid is separable by extraction with an aqueous alkaline solution [33,34]. Also, fiuorous phase separation techniques have been combined with enzymatic kinetic resolutions [35]. To overcome the 50% yield limitation, one of the enantiomers may, in some cases, be racemized and resubmitted to the resolution procedure. 

The combination of chromatography and mass spectrometry (MS) is a subject that has attracted much interest over the last forty years or so. The combination of gas chromatography (GC) with mass spectrometry (GC-MS) was first reported in 1958 and made available commercially in 1967. Since then, it has become increasingly utilized and is probably the most widely used hyphenated or tandem technique, as such combinations are often known. The acceptance of GC-MS as a routine technique has in no small part been due to the fact that interfaces have been available for both packed and capillary columns which allow the vast majority of compounds amenable to separation by gas chromatography to be transferred efficiently to the mass spectrometer. Compounds amenable to analysis by GC need to be both volatile, at the temperatures used to achieve separation, and thermally stable, i.e. the same requirements needed to produce mass spectra from an analyte using either electron (El) or chemical ionization (Cl) (see Chapter 3). In simple terms, therefore, virtually all compounds that pass through a GC column can be ionized and the full analytical capabilities of the mass spectrometer utilized. 

The characteristics of an ideal liquid chromatography-mass spectrometry interface have been discussed, with emphasis having been placed upon the major incompatibilities of the two component techniques that need to be overcome to allow the combination to function effectively. 

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