Functional analysis HPLC separation

The analysis of a technical poly(ethylene oxide) with respect to chemical composition and degree of polymerization has been performed by Pasch and Hiller [210]. This investigation was conducted under conditions which are common for HPLC separations, i.e. sufficiently high flow rate, moderate sample com-centration, and on-flow detection. Using an octadecyl-modified silica gel as the stationary phase and an eluent of acetonitrile/deuterium oxide 50 50 (v/v), the sample was separated into different functionality fractions (see Fig. 38). The major fraction of the sample eluting between 14 and 25 min exhibited a partial oligomer separation. 

Figure A16.1 HPLC separation of components in a mixture. The chromatograms are from Ying Liu et al.. Behavioral and Brain Functions 3 47 (2009) doi.1186/1744-9081-3-47. They show analysis of morning urine samples from a normal subject (top) and a severely depressed female patient (below). The analysis is looking for significant differences in the content of peptides in the urine. The samples in such analysis are injected into the HPLC column and as the samples passes down the column the components separate so that they emerge (left to right) as separate sharp peaks, which can be detected by their u.v. light absorption properties. The difference between the normal subject and the patient is striking.

The range of most convenient hydrophobicity of organic compounds for reversed-phase (RP) HPLC separation may be estimated approximately as —1 < log P < + 5 (log P is the logarithm of the partition coefficient of the compound being characterized in the standard solvent system 1-octanol/water). Highly hydrophUic substances with log P < —1 need a special choice of analysis conditions, e.g., introduction of ion-pair additives into the eluents. Another approach is their conversion to more hydrophobic derivatives by the modification of functional groups with active hydrogen atoms. 

High performance spectroscopic methods, like FT-IR and NIR spectrometry and Raman spectroscopy are widely applied to identify non-destructively the specific fingerprint of an extract or check the stability of pure molecules or mixtures by the recognition of different functional groups. Generally, the infrared techniques are more frequently applied in food colorant analysis, as recently reviewed. Mass spectrometry is used as well, either coupled to HPLC for the detection of separated molecules or for the identification of a fingerprint based on fragmentation patterns. ... 

This separation technique produces very good results for acidic or anionic dye molecules containing carboxylic, sulfonic, and hydroxy groups that can be separated within short run times in an aLkaline medium in a single analysis step. - Natural colorants usually do not contain these functional groups they are usually more voluminous and strongly hydrophobic, properties that complicate their determination by CE. The sample pretreatment is more difQcult when CE (compared to HPLC) is used. 

The area under a chromatographic peak is proportional to the concentration of the corresponding.species in the sample analysed. Using empirical calibration functions, a rapid simultaneous qualitative and quantitative analysis of sulfur mixtures by HPLC is now possible. In a similar manner the selenium rings Seg, Se, and SCg have recently been separated by HPLC... 

The marriage of HPLC to mass spectrometry (MS), now developed into a mature instrumentation, continues to greatly impact many of the separation sciences, especially in pharmaceutical analysis where it has been used in new drug discovery [23,24] and in drug metabolite identification [25-27]. HPLC-MS has also made an impact on lipid research, providing a convenient approach to the analysis of phospholipids and fatty acids [28,29]. It has also greatly benefited the field of proteomics [30-34], especially analysis of protein structure and function. 

The application of high-performance capillary zone electrophoresis (HP-CZE) in its various selectivity modes has become a very valuable adjunct to HPLC for the analysis of peptides. For synthetic peptides, in particular, both HPLC and HP-CZE now form essential components of the analytical characterization of these molecules. Increasingly, zonal, micellar, or (biospecific) affinity-based HP-CZE procedures with open tubular capillary systems are adapted to allow resolution with extremely high separation efficiencies (e.g., >105 plates per meter) of synthetic or naturally occurring peptides as part of the determination of their structural, biophysical, or functional properties. Illustrative of these capabilities are the results shown in Figure 19 for the separation of several peptides with different charge and Stokes radius characteristics by HP-CZE. 

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