Sample capacity HPLC development

For biological samples where the analytes often are non-volatile and/or occur in an aqueous matrix, the reversed-phase mode, using a hydrophobic stationary phase and an aqueous mobile phase is extremely useful. The usefulness and popularity of HPLC was further increased by the possibility to automate and computerize the systems providing unattended operations and high sample capacities. Many Nobel Prize awards have been based upon work in which chromatography played an important role [5], Most recently, the 2002 Nobel Prize in chemistry was awarded to the development of methods for identification and structure analyses of biological macromolecules" in which HPLC and Mass spectrometry (MS) were used [6],... 

An interesting development is the combination of HPLC and on-line measurement of reducing capacity or antioxidative activity. This approach allows both direct identification of antioxidative species in complex foods and quantification of the contribution to the overall antioxidative capacity in the absence of synergistic and antagonistic effects. Major advantages are less sample handling and the ability to rim large series of samples in an automated process. 

For the analysis of nonvolatile compounds, on-line coupled microcolumn SEC-PyGC has been described [979]. Alternatively, on-line p,SEC coupled to a conventional-size LC system can be used for separation and quantitative determination of compounds, in which volatility may not allow analysis via capillary GC [976]. An automated SEC-gradient HPLC flow system for polymer analysis has been developed [980]. The high sample loading capacity available in SEC makes it an attractive technique for intermediate sample cleanup [981] prior to a more sensitive RPLC technique. Hence, this intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest. Coupling of SEC to RPLC is expected to benefit from the miniaturised approach in the first dimension (no broadening). Development of the first separation step in SEC-HPLC is usually quite short, unless problems are encountered with sample/column compatibility. 

The fabrication of imprinted monolithic solid-phase microextraction fibres has been developed for the selective extraction and preconcentration of diacetylmorphine and its structural analogues, triazines, bisphenol A, anaesthetics, and antibiotics followed by GC or HPLC analysis [156,163,179,196,197]. In addition, the on-line coupling of the imprinted monolith as a preconcentration column with a conventional analytical column has been proposed for the enrichment and cleanup of environmental and food samples [163]. However, at present, the capacity of the imprinted fibres and thus the degree of recovery of analytes are very variable and obviously need some improvement. For example, the recoveries of triazines after SPME with an imprinted monolith prepared by in situ polymerisation of MAA as... 

The scale of preparative HPLC is normally larger than that of conventional HPLC. Therefore, a practical starting point is to develop an analytical separation that optimizes the isolation conditions. Optimization of the analytical method implies seeking conditions that combine maximum resolution of the peak of interest and minimum elution time, under the restriction of a limited pressure drop. The optimized conditions determine the column, mobile phase, flow rate, and sample loading capacity for the particular column. The conditions may be either normal phase or reverse phase. The mobile phase should be chosen carefully to avoid salt complexation with the compound to be isolated. Volatile acid salts such as trifluoroacetic acid, formic acid, and acetic acid are acceptable mobile phase additives, and the ammonium counterion is preferred for pH adjustment to any of these acids. 

The development of the first HPLC system with MW-triggered fraction collection was described by Zeng et al. [50] at CombiChem. This system was a dual analytical-preparative instrument with parallel-column format, termed parallel Analyl/PrepLCMS." developed by the modification of commercially available instrumentation. This system had software-controlled valves that applied sample to each path from a single autosampler and had the capacity to purify and analyze more than 100 samples per day. Initial analytical LC-MS data acquired by the system allowed the identification of samples that require... 

Reversed-phase HPLC is the method of choice for the quantitative analysis of procyanidins. In view of the known instability of procyanidins and the problem of developing suitable sample clean up procedures direct analysis of crude extracts is probably the best approach for quantitation. However, the separation capacity of HPLC in combination with the most commonly used UV detection at 280 nm is generally insufficient to generate useful quantitative results. Direct chromatographic determination of procyanidins in qualitative analysis has been frequently performed for example in the analysis of wine [168,252], beer [32], grape seeds [28], rhizomes of tormentil (Potentilla tormentilla) [253], Sesbania sesban... 

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