Resolution, fractionation techniques

In homopolymer analysis this meant a closer study of the accuracy and reproducibility of data from GPC to see how resolution correction techniques could be either circumvented or practically applied. In copolymer analysis the limitation of conventional molecular size fractionation emerged as the fundamental difficulty. An orthogonal coupling of GPCs operated so as to achieve the desired cross fractionation before detection is presented as a novel approach with considerable potential. 

Because of the complex nature of most biological samples, a single fractionation technique may not be adequate for the separation of the wide range of molecules present. Better resolution of some molecules is obtainal when properties other than differences in size are exploited. These include differences in ionic characteristics, affinity for other molecules and hydrophobicity. In separations that involve any one or more of these properties, the sample constituents interact with the column material and are then eluted with a suitable eluant. As a consequence of this interaction, and the use of eluants, whose properties may not closely resemble those of the medium found in vivo, the metal may dissociate from the ligand. In addition, as the complexity of the sample increases it is difficult to predict the behaviour of the various constituents. Undesirable effects leading to irreversible interaction between some molecules in the sample and the column packing material, degradation and decomposition of some constituents may result. Furthermore, it may be difficult to rid the column of certain trace metal contamination. 

Polybrominated Diphenyl Ethers. Like PCBs, air samples containing PBDEs are usually collected by pumping air through a sampler containing a glass fiber filter and adsorbent trap to separate the particle bound and vapor phase fractions, respectively (Dobber et al 2000a Hillery et al 1997). The filters and adsorbants are then Soxhlet extracted with acetone/hexane, and the extracts are cleaned-up and analyzed by high resolution GC techniques. 

The determination of polyphenolics may result in interference due to co-elution of phenolic acids and procyanidins. This problem can be eliminated by fractionation of polyphenolics into acidic and neutral polyphenolics prior to sample injection into the HPLC system. Because the fractionation techniques effectively improve the resolution of many polyphenolic peaks in the reversed-phase HPLC system, it is suggested that further characterization and identification of unknown peaks be conducted by additional methods such as mass spectrometry and nuclear magnetic resonance. 

Biological systems are a complex mixture of proteins, peptides, amino acids, inorganic complexes and ions. Because the systems are so complex it is virtually impossible to use a single fractionation technique to resolve the various constituents. However, complete resolution of the constituents is not a prerequisite in the study of trace metal bound fractions. Indeed, the extent to which the constituents are to be resolved by the separation technique is determined primarily by the selectivity and sensitivity of the available detection techniques. 

A fractionation technique with low resolution coupled with a highly selective detection may be more powerful than a high resolution separation coupled with a relatively non-se-lective detection. In this regard, techniques that can be used specifically to determine the metal or a class of ligands are preferred to those that measure properties like the UV spectra or refractive index. Only in cases where the metal is associated with two partly unresolved species is the use of two or more fractionation techniques justified. It must be emphasised at this point that the chances of disrupting the metal-ligand association increase as the number of fractionation steps increase. 

One further point to note is that comparing the NMR results with the GLC traces for the same fractions it is noticed that the NMR peaks appear to agree quantitatively as well as qualitatively with the GLC peaks. This suggests that the high resolution NMR technique may be useful for analysing the smaller cyclic oligomers (up to and including 15 repeat units). 

---