Elution methods development

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

With only 100 pg total protein loaded (for method development), peaks I and II were very well resolved. When the full sample (6 mg) was injected for preparative purposes, peak II shifted to an earlier retention time. A shift to earlier retention on increased loading is a common problem in purification. If the major component can be made to elute before the minor component, the retention shift will not harm the separation as greatly as if the major component elutes after the major component. 

Column Selection The selection of the two types of columns to be used is perhaps the most important consideration in 2DLC method development. This is driven by the need to have orthogonal dimensions for the solutes under investigation, otherwise the solutes will elute along the diagonal of the separation space, as discussed in Chapter 2. We have observed a number of 2DLC applications in the literature,... 

In complex samples, when the range of elution times may not be known beforehand, there is the possibility of wraparound where components from the previous run are still eluting on the next second-dimension elution (Micyus et al., 2005). This situation is of concern and should be eliminated in the method development process for all but the most exploratory of work. This may require collecting fractions and injecting these fractions into the second-dimension column to determine the most retained compound retention time as part of the method development process. 

The method development process with the multisorbent plate consists of three steps. In step 1, the sorbent chemistry and the pH for loading, washing, and elution are optimized. In step 2, optimization of the percentage organic for wash and elution and the pH of the buffer needed is carried out. Step 3 is validation the method developed from the results of the previous two steps is tested for linearity, limits of detection, quantitation of recovery, and matrix effects using a stable isotope-labeled analyte as an IS. 

FIG U RE 1.19 (A) Load-elution study on moderately hydrophobic, neutral carbamazepine with a four-sorbent method development plate. (B) Load-elution study of procainamide with multisorbent method development plate. (C) Load-elution study of acidic indomethacin with multisorbent method development plate. 

In addition, the use of fast gradients elution mode has become the bioanalytical mainstream as a possible way to improve peak parameters (shape and symmetry) and to minimize method development time, especially for the multi-analytes methods. 

In a similar manner, NP conditions (chloroform-based eluents) could be adopted for the separation of iV-tert-butoxycarbonyl-proline (Boc-Pro) enantiomers on a hybrid urea-linked epiquinine-calixarene type CSP and an acidic displacer such as acetic acid promoted elution [42]. Since Ink vs. ln[CH3COOH] dependencies gave straight lines, it may be concluded that this may be attributed to an ion-exchange process still existing in the NP mode. Acids cannot be eluted within reasonable run time without adding an acidic displacer. The practical relevance of the NP mode has to be seen in its much wider solvent choice, which may greatly extend the fiexibility in the course of method development. 

A PDA detector provides UV spectra of eluting peaks in addition to monitoring the absorbance of the HPLC eluent like the UVA is absorbance detector. It is the preferred detector for testing impurities and for method development. PDA facilitates peak identification during methods development and peak purity evaluation during method validation. Detector sensitivity was an issue in earlier models but has improved significantly (more than ten-fold) in recent years. ... 

---