Large versus Small Analyte Molecules and Their Derivatizations

It is generally easier to derivatize small molecules than large ones, since the rates of chemical reactions of very large biomolecules are usually orders of magnitude slower than those of smaller species. This is a function of effective chemical collisions, the number of chemical collisions per unit time between reactive sites, conformational preferences of large biomolecules, and the number of active sites available in a biomolecule. That is not to say that biomolecules cannot be successfully derivatized - they often are and can be - but the efficiency of derivatization (percent derivatization per unit time) versus smaller reactive species is usually much less. Also, the energy of activation needed to derivatize a primary amino group in a large molecule is often much larger than that for a very small molecule having the same functionality. This is, of course, a function of the neighbouring groups, conformational preferences, conformations available, hydrogen bonding within the biomolecule, and other factors. A considerable danger with derivatizing large molecules (typically biopolymers) stems from the fact that, in most cases, such a polymer possesses a number of reactive groups, for reasons just specified, which may differ in their reactivity. The result may be the formation of a number of products bearing the same tag in different mole per mole ratios. Although in enzymatic amplification techniques the formation of multiple products helps identification, in the situation just described the formation of multiple derivatization products should be avoided. The separation of such mixtures is often difficult, usually resulting in broad peaks and low plate counts. Moreover, it may be difficult to trace back which derivative was derived from which solute present in the original sample.

Numerous chemical reactions have been used to derivatize different classes of biomolecules in LC, usually with a high degree of success. However, the overall enhancement is always dependent on the particular tags used. That is, derivatization reactions that tag a specific site within the biomolecule sometimes lead to a single, and sometimes several, tags incorporated into the derivative. As a function of the tag, there will be improved detector response, but perhaps much smaller chromatographic changes than with small molecules if the derivatization is carried out pre-column. Derivatizations are therefore often performed post-column. An ideal derivatization scheme would generate many derivatives from the original biomolecule, such as via enzyme amplification. This is already used to detect intact enzymes, but is used much less to detect proteins, peptides, nucleic acids, etc. Thus, the scheme described using post-column, microwave digestion of proteins, followed by a second post-column solution reaction with a FL derivatizing reagent (e.g. o-phthaldialdehyde, OPA), leads to many amino acids now detectable by FL methods. This is, perhaps, an ideal example of a general approach that greatly improves detectability of large molecules, such as via enzyme amplification for enzymes.

Solar Panel Basics

Solar Panel Basics

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