Isocratic focusing

Isocratic focusing techniques, in conjunction with turbulent-flow chromatography, are also explored in this chapter. Isocratic focusing is used to improve chromatographic peak shape, enhance sensitivity, and optimize reproducibility. Isocratic focusing has also led to the development of generic methodologies that eliminate the need... 

Figure 10.6. Isocratic focusing method configuration (a) sample cleanup (b) sample transfer (c) sample elution.

Figure 10.7. Comparison of 100ng/mL K252a (a) no isocratic focusing (b) isocratic focusing...

TABLE 10.3. Comparison of 10 Replicate Injections of 100 ng/mL K252a over Time with and without Isocratic Focusing (Herman, 2002). 

TABLE 10.4. Selected Applications Using Isocratic Focusing TFLC and LC-MS/MS Detection... 

Figure 10.9. 100ng/mL K252a after 1000 injections of plasma (a) without isocratic focusing (b) with isocratic focusing (Herman, 2002). 

Isocratic focusing can also be used to concentrate analytes online (Herman, 2005). Analytes from several injections can be extracted, cleaned, and transferred to the HPLC column before performing the gradient elution step. Analytes will be retained on the analytical column because the column is only exposed to a high aqueous mobile phase during the clean and transfer steps. The gradient elution step is performed once the appropriate number of injections has been concentrated. This methodology is quite useful for low-level metabolite identification (Herman, 2005). 

The general methodology for online cleanup and concentration is the same as that used for isocratic focusing when assaying for known compounds, but sample cleanup and transfer steps are repeated as many times as necessary to concentrate the sample. Once the concentration is complete, elution from the analytical column occurs. LC-MS responses of a single standard injection to one that has been concentrated 10 times (10 injections) are compared in Fig. 10.12. Note that the response remains linear as long as the trapping analytical column is not over loaded. 

Figure 10.12. Comparison of 10ng/mL standard in rat plasma (a) single injection vs. (b) 10 injections concentrated online with isocratic focusing (Herman, 2005).

Isocratic focusing effects are used to (1) enhance peak shape (2) allow generic approaches to rapid pharmacokinetic screening, (3) perform online concentration for low level metabolite identification, and (4) provide for reducing the amount of... 

Herman, J. L. (2005). The use of turbulent-flow chromatography and the isocratic focusing effect to achieve on-line cleanup and concentration of neat biological samples for low-level metabolite analysis. Rapid Commun. Mass Spectrom. 19 696-700. 

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