Biopolymer analysis

In biological specimens, the major biopolymers, that is, proteins, nucleic acids, lipids and polysaccharides are frequently encountered in close association with each other or with other cell components. Well-established procedures are, however, available for their removal and separation from isolated cell fractions. 

The complete analysis and sequence determination of proteins and nucleic acids relies on the splitting of these high molecular weight compounds into smaller fragments, using chemical and/or enzymatic methods. The smaller peptide or oligonucleotide fragments so produced are then analysed by various chemical or physical methods. 

The enormous advances in techniques which have been made during the last decade or so, have enabled the amino acid sequences of many proteins, and the nucleotide sequences of many nucleic acids to be worked out. Complimentary XRD work with crystalline specimens has elucidated many three-dimensional features of secondary and tertiary structure. There has been general confirmation of the a-helix protein structure originally proposed by Pauling, Corey and Branson [28] and the double helix idea of DNA structure conceived by Crick and Watson [29]. 

Nucleoprotein complexes in biological specimens can frequently be separated into their components by fairly simple methods. In the presence of concentrated phenol and a detergent, for example, a cell homogenate will form two liquid phases. Proteins are denatured and become insoluble in the aqueous phase, while the nucleic acids remain soluble. Alternatively, the separation of protein and nucleic acid components from an aqueous NaCl solution can be effected with chloroform (Chapter 11.4). 

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Due to recent advances in column technology, novel stationary phases have become available for such applications. This communication deals with the use of micropellicular sorbents which consist of a fluid-impervious microspherical support with a thin retentive layer at the surface. For biopolymer analysis by HPLC, such stationary... 

In the manufacture of proteinaceous drugs, the purity of the final product is of paramount importance. Purity, however, is by no means an absolute term it depends on the method used for its measurement. Since traditional chromatographic methods and slab gel electrophoresis have been the main tools for biopolymer analysis, the commonly used but utterly vague terms chromatographicalfy or electrophoreticalfy pure demonstrate the role of available techniques not only in the measurement but also in the definition of purity. 

The advantages of HPLC over classical chromatographic methods stem from the employment of a precision instrument that utilizes high-performance columns with concomitantly high analytical speed and resolution and affords total control over the chromatographic process and sensitivity of analysis. In a way, the recent emergence of capillary electrophoresis (CE) follows the same patterns electrophoresis, a well-established and widely used method of biopolymer analysis, is carried out... 

Most of the pyrolysis experiments in the field of natural polymer characterization involve the use of Curie-point instruments or resistance-heating apparatus. Both methods are described in the following sections. Laser pyrolyzers are not yet common in biopolymer analysis and will not be discussed here. 

High performance capillary electrophoresis is one form of free-solution electrophoresis. CE is useful to researchers and analysts working in areas in which traditional electrophoresis is customarily applied (e.g., biopolymer analysis) and also in disciplines not usually associated with electrophoretic analysis, such as inorganic ion analysis. The potential application areas of CE are vast, because this technique can separate a variety of ligates, from inorganic ions up to intact cells, using the same instrumental hardware designed for separations based on different physical-chemical mechanisms. 

In order to improve the separation efficiency and speed in biopolymer analysis a variety of new packing materials have been developed. These developments aim at reducing the effect of slow diffusion between mobile and stationary phase, which is important in the analysis of macromolecules due to their slow diffusion properties. Perfusion phases [13] are produced from highly cross-linked styrene-divinylbenzene copolymers with two types of pores through-pores with a diameter of 600-800 mu and diffusion pores of 80-150 nm. Both the internal and the external surface is covered with the chemically bonded stationary phase. The improved efficiency and separation speed result from the fact that the biopolymers do not have to enter the particles by diffusion only, but are transported into the through-pores by mobile-phase flow. 

Another important development in the field of biopolymer analysis is the introduction of matrix-assisted laser desorption ionization (MALDl), which is a rather recent soft ionization technique that produces molecular ions of large organic molecules. In combination with time-of-flight (TOP) mass spectrometry, it was proposed as a valuable tool for the detection and characterization of biopolymers, such as proteins, peptides, and oligosaccharides, in many types of samples.The use of these recently developed techniques has not decreased the use of chromatography in determinations of biopolymers. Some efforts on the adaptation of the separation abilities of HPLC to the high potential of MALDl-TOF for the sensitive determination of additives in biocomposites are currently being carried out. 

In all these applications, the separation step is one of the most critical during the whole analytical process. Solid phase extraction (SPE) and capillary electrophoresis (CE) were also proposed for high-resolution and quantitative separations of analytes. Therefore it is likely that the use of chromatographic techniques in this area will be increased in the near future. The development of adequate interfaces for such hyphenated techniques is the most important problem to be solved by researchers in the field of biopolymer analysis. 

The attempts that have been made to utilize true chemometric optimization of operating conditions in CEC are unclear in most of the studies done utilizing CEC. This has been done for many years in GC and HPLC, as well as in CE, but there are no obvious articles that have appeared which have utilized true chemometric software approaches to optimization in CEC [57-59]. It is not clear that any true method optimization has been performed or what analytical figures of merit were used to define an optimized set of conditions for biopolymer analysis by CEC. It is also unclear as to why a specific stationary phase (packing) was finally selected as the optimal support in these particular CEC applications for biopolymers. In the future, it is hoped that more sophisticated optimization routines, especially computerized chemometrics (expert systems, theoretical software, or simplex/optiplex routines) will be employed from start to finish. 

Sometimes the sample preparation is a difficult problem, especially in clinical and environmental chemistry. General procedures are filtration (perhaps by means of a dedicated membrane which retains compounds selectively), solid phase extraction with disposable cartridges (also with dedicated selectivity), protein precipitation and desalting. A special case is sample preparation for biopolymer analysis. 

The type and quality of solvents can influence the MALDI analysis of polymer samples. For example, the dryness and purity of tetrahydrofuran (THF) used to prepare polymer samples play a central role in the success of detecting high-molecular mass polymers [29]. The solvent system used can affect analyte incorporation and distribution in matrix crystals. As has been shown in MALDI biopolymer analysis, analyte distribution in matrix crystals can significantly affect the signal reproducibility, detection sensitivity, and relative intensities of individual components in a mixture [40]. However, unlike biopolymer analysis-where a... 

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