Gradient elution ideal system

The transport detector is ideal for most gradient elution applications, the major limitation being those which use involatile buffers. The full potential of the FID cannot be realised due to the deficiencies of the transport system. The overall detector response is dependent on temperature stability and on the coating procedure which is related to both viscosity and surface tension of the solvent and sample. Due to these drawbacks particularly the lack of sensitivity these detectors were soon withdrawn. 

Chemical modification of saccharides is an ideal tool for shifting their absorption maximum into a range which facilitates classical UV detection but suffers from inherent problems (Section 11.4.4) which precludes the popular use of this methodology. Detection of changes in refractive index is the preferred method of detection of the saccharides and detection limits are in the range 10-40 fig and depend upon the specific system employed. More recently mass detectors have been described which can be used with gradient elution and which achieve a 10 times greater sensitivity than refractive index detectors (Macrae and Dick, 1981). Less common techniques for the... 

Measurement of labelling yield and subsequent radiochemical purity requires a suitable analytical technique, and the method of choice for radio-labelled peptides is reversed phase HPLC with on-line UV and radiometric detection. It is important to use as stringent a separation method as possible with isocratic or slow mobile phase composition gradients over the peptide peak. Ideally, more than one mobile phase system should be used (e.g. a phosphate buffer-methanol system in addition to the standard water-acetonitrile system), since these may show the presence of new impurities. It is important to recognize that HPLC analyses only measure those components that elute from the column. Insoluble, highly lipophilic or positively charged species may bind to the solid phase. It is very important to verify the absence of these species by a complimentary technique such as thin layer chromatography (TLC) and to ensure that the two techniques produce similar results. 

Advances in stationary phase technology have led to commercial availability of adsorbents such as high performance sihcas, aluminas, polyamides, celluloses and derivatised silicas [9,10], The development of automated method development (AMD) systems [127] now allow multi-step gradients of different elution strengths to be achieved in a relatively short time compared to earlier manual approaches. AMD systems are ideally suited for separation of complex mixtures with a wide range of polarities. Further improvements in sample resolution and reduced method development times in TLC include the use of two-dimensional development approaches [128] and forced-flow development by over-pressure liquid chromatography (OPLC) [129]. 

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