Gradient-elution method development

Weller et al. [43] described an early high throughput UV-triggered HPLC purification system to support the parallel synthesis efforts at Bristol-Myers Squibb. This open-access instrument used fast flow rates and rapid universal reverse-phase gradient elution methods that enabled the purification of up to 200 samples per day at weights up to 200 mg/sample in an unattended mode. Customized software and hardware were developed for this system to optimize efficiency and throughput. 

The fuU mathematical treatment of gradient elution (Snyder 1979) that led to these relationships (Table 3.2) is too complex to be reproduced here indeed, in practice a gradient elution method is often developed by an essentially trial-and-error approach based on an isocratic method while keeping in mind the restrictions imposed by the qualitative considerations outlined above. However, it is possible to give some flavor of this theory to provide some understanding of the principles, as follows. 

The use of a two-coliunn technique in separating this mixture can be avoided if gradient elution methods are applied. They are accomplished by means of a device which increases the polarity of the developing sol-... 

Therefore, if a fast, automated, routine method for the measurement of multi-species/elements is the desired analytical goal, it is often best to attempt an isocratic separation method first, because of the complexity of method development and the low sample throughput of gradient elution methods. In fact, a simultaneous method for the separation of As, Cr, and Se species in drinking water samples was demonstrated by Neubauer and coworkers they developed a method to determine inorganic forms of arsenic (As, As+ ), chromium (Cr , Cr ), and selenium (Se, Se and SeCN ) by reverse-phase ion-pairing chromatography with isocratic elution. Details of the HPLC separation parameters/conditions they used are shown in Table 18.2. 

Thin-layer chromatography (TLC) is used both for characterization of alcohol sulfates and alcohol ether sulfates and for their analysis in mixtures. This technique, combined with the use of scanning densitometers, is a quantitative analytical method. TLC is preferred to HPLC in this case as anionic surfactants do not contain strong chromophores and the refractive index detector is of low sensitivity and not suitable for gradient elution. A recent development in HPLC detector technology, the evaporative light-scattering detector, will probably overcome these sensitivity problems. 

Selectivity of the separation in TLC is achieved by various of the aforementioned techniques (e.g. multiple development, gradient elution, sequence TLC, AMD, HPPLC or OPLC). Multidimensional TLC methods are described in Section 7.4.4. 

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