Amino Acid and Peptide Analytes

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

Jorgenson reported the use of glass capillaries for free solution electrophoresis 25 years ago (Jorgenson andLukacs, 1981,1983). Aplug of analyte was introduced into a buffer-filled capillary and separated at high electric fields. Capillaries of 75 im inner diameter were employed, and detection of labeled amino acids and peptides was based on fluorescence. 

Displacement chromatography was named and introduced by Tiselius [1] who demonstrated its utility in both preparative and analytical applications. Tiselius explored separations of many different biological substances including amino acids and peptides [2,3]. The technique was also used... 

Although the majority of reports of macrocycles in analytical chromatography have involved ligand association with the stationary phase, their use as mobile phase constituents has also been investigated. Lamb and Drake [11] showed that addition of water-soluble crown ethers to the mobile phase altered the retention of alkali metal cations on an underivatized reversed phase column. Nakagawa et al. [63-66] also used crown ether-containing mobile phases in the separation of protonated amines, amino acids and peptides, and [1-lactam antibiotics. 

Analytical Properties Separation of compounds containing the NH4+ group, such as amino acids and peptides the coated silica also behaves as a reverse phase for the separation of aliphatic and aromatic acids high selectivity for glycine and tyrosine oligomers Reference 59... 

One constant problem for crown ether based sensors in vivo is the ubiquitous presence of chemical species that will compete with the target analyte for the sensor binding site. For sensors incorporating [18]crown-6 this especially problematic as sodium, potassium, ammonium and hydronium (H30+) cations are all attracted to the threefold symmetry of the crown s cavity. Protonated terminal amines, including amino acids and peptides, can also interfere with analyte detection. It is therefore all the more pleasing when a crown ether based sensor is developed that does not bind to biologically common cations. This is the case for a saxitoxin chemosensor reported by Gawley, LeBlanc and co-workers [24],... 

Knapp, D. (1979). Derivatization of amino acids and peptides. Handbook of Analytical Derivatiza-tion. Wiley-Interscience, New York. pp. 243—302. 

In this context, a major impact has been the development of high-resolution reversed phase chromatographic (RP-HPLC) techniques for analytical and preparative purposes.t l In fact, with the discovery of reversed stationary phases and with applications to chromatographic resolution of amino acid and peptide derivatives,HPLC very soon emerged as the most powerful technique for synthetic peptides.P l... 

His published work covered many fields and included diverse subjects such as the chemistry of amino acids and peptides, especially glutathione mineral metabolism, with special reference to calcium melanin pigment metabolism ascorbic acid metabolism metabolic aspects of cardiac muscle and analytical techniques for lipids, nitrogenous compounds, and cortisol. He was an extraordinarily meticulous analyst who, from the first, maintained that the standards of technique in the service laboratory should be the same as those required for research purposes. He maintained that the fulfillment of clinical chemistry demanded equal collaboration between physician and chemist. The function of the latter was not to usurp that of the former but to assist the clinician by helping to shed light on the nature of an illness. 

Reactions and analytical methods for amino acids and peptides... 

Part 1 of this chapter is intended to provide background material for the analytical procedures described later in this chapter for amino acids and peptides, but it also provides a broad survey of the topic that can be read in isolation from the analytical context. The derivatisation of amino acids is the basis of many of the sensitive analytical amino-acid assay procedures in current use and this chapter covers the normal profile of reactions of the amino and carboxy groups, knowledge of which is an essential prerequisite for appreciating the analytical context. Reactions of peptides are also covered here (e.g. peptide and protein hydrolysis is covered in Section 4.4.7), though the coverage is restricted in scope because parts of this topic are discussed in Chapter 5, where it is relevant to sequence-determination procedures (see also Barrett, 1985). 

Thin-layer chromatography (TLC) provides convenient routine analytical support of synthesis and other amino-acid interests and has been used in the Mosher procedure just described. It is most generally used for free amino acids and peptides, with spray reagents based on ninhydrin, or on the above derivatives ( post-TLC derivatisation ). Dansyl and phenylthiohydantoin (PTH) derivatives have been used for many years for identifying amino acids in mixtures by TLC ( pre-TLC... 

The two purposes of separation of amino-acid and peptide mixtures are either at the preparative level, to isolate one or more individual components from the mixture for further study or at the analytical level, to identify and to determine the relative amounts of some or all of the components. Most of the routine studies, conducted daily to determine the amino-acid content of clinical and botanical samples in hundreds of laboratories around the world, are at the analytical level. However, many of the research studies are at the preparative level an example of this is the identification of crosslinking amino acids from proteins, through their isolation from protein hydrolysates, from physiological specimens for medical investigations, or purely to gain new knowledge. 

The scheme summarises all modern analytical and preparative chromatography protocols, such as high-performance liquid chromatography (HPLC) and gas-liquid chromatography (GLC), with all their conceivable variations. Reverse-phase HPLC or GLC , in which a non-polar liquid is adsorbed onto the solid - the stationary phase - is more appropriate for the analysis of mixtures of derivatives of amino acids and peptides. Cellulose in the above scheme would be replaced by a less-polar medium, such as acetylated cellulose, silanised silica gel, etc. in standard reversed-phase HPLC. 

Sufficient volatility for GLC analysis is found for A-acylated esters of amino acids and peptides. Their preparation requires a two-step derivatisation protocol and therefore introduces a potential source of error. There is also anxiety about the impurities that may be introduced in this way. However, this applies to any derivatisation protocol and experienced users of the GLC technique can obtain impressive reproducibility of results, sufficient to match the reliability of the classical Moore and Stein procedure. Flexibility because of the additional range of detectors available for GLC can be useful, e.g. highly sensitive electron-capture detectors for halogenated analytes or amino acids and peptides derivatised with halogen-containing groups. 

This is an undergraduate and introductory postgraduate textbook that gives information on amino acids and peptides, and is intended to be self-sufficient in all the organic and analytical chemistry fundamentals. It is aimed at students of chemistry, and allied areas. Suggestions for supplementary reading are provided, so that topic areas that are not covered in depth in this book may be followed up by readers with particular study interests. 

Amino acids and peptides are easily labeled with fluorescent dyes. CZE, CGE, or MEKC in capillary or microchip-based SCCE can typically resolve these analytes. Microchip-based SCCE offers higher sensitivity for very small samples, and higher electric field strengths can be used without Joule heating problems. Capillary-based SCCE offers a much higher peak capacity and the use of UV absorbance detection of unlabeled analytes. 

In New York, Bergmann continued his investigations of amino acids, peptides and proteins on a wider base. This was done through intensive work on analytical methods for the determination of amino acids and peptides and also through an enhanced examination of protein-cleaving enzymes with the necessary synthesis of peptides suitable for serving as model substrates. 

In early mass spectrometry applications of lasers, the sample was irradiated directly by a laser beam to desorb intact sample-related ions [27]. In this direct mode, termed laser desorption/ionization (LDI), the extent of energy transfer is, however, difficult to control and often leads to excessive thermal degradation. Also, not all compounds absorb radiation at the laser wavelength and thus are not amenable to LDI. Only those compounds that have mass below 1000 Da can be analyzed by LDI. Analytical sensitivity is also poor. A key contribution of LDI experiments is the observation that the desorption efficiency of amino acids and peptides that absorb the laser fight beam is greater than those without the chromophore [28]. IR lasers (e.g., an Nd YAG laser at 1.06 p m and a pulsed CO2 laser at 10.6 pm) and UV lasers (frequency-quadrapled Nd YAG laser at 266 nm) have aU been used. The detection of malaria parasites in blood by LDI with an N2 laser has been demonstrated [29]. 

The influence of micelles on the acid-base properties of analytes can be significant and is reflected in changes in log Kh. A gocd example is the acid-base behavior of amino acids and peptides, which present the following equilibria [44,45] ... 

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