Method Development RPLC

Once a suitable measurement technique has been selected, a process of method development must be initiated. Within our laboratories, for RPLC, we employ a screening process to get to the most robust separation conditions in the shortest amount of time. The following details each step 

With the ultimate objective of maximum method robustness and ruggedness in mind, selection of the appropriate stationary phase is 

FIGURE 16.13 Example plot of log D versus mobile phase pH for a pharmaceutical 

In those cases, secondary column screens or alternative elution modes (SFC, HILIC) are applied. 

Stationary phase Organic modifier Aqueous buffer/pH 

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TABLE 16.2 16.3. CHROMATOGRAPHIC METHOD DEVELOPMENT RPLC 449 Example of the Second Screening Sequence Used to Optimhe the Temperature and Gradient Elution ... 

Applications Off-line SFE-HPLC appears to be applicable and quantitative for a variety of samples in many real -world matrices. The main challenge lies in the use of this technique for the more polar compounds. Quantitative off-line SFE-SFC-UV analysis of HDPE/Ethanox 330 was described after extensive method development (varying modifiers, modifier concentration, temperature) [129]. Soxhlet extraction and SFE-RPLC-UV of PE samples were compared [127]. A sample size (inhomogeneity) problem was pointed out when a SFE reproducibility study was performed on five 3-mg samples of PE. This points to limits... 

Others have examined the necessary parameters that should be optimized to make the two-dimensional separation operate within the context of the columns that are chosen for the unique separation applications that are being developed. This is true for most of the applications shown in this book. However, one of the common themes here is that it is often necessary to slow down the first-dimension separation system in a 2DLC system. If one does not slow down the first dimension, another approach is to speed up the second dimension so that the whole analysis is not gated by the time of the second dimension. Recently, this has been the motivation behind the very fast second-dimension systems, such as Carr and coworker s fast gradient reversed-phase liquid chromatography (RPLC) second dimension systems, which operate at elevated temperatures (Stoll et al., 2006, 2007). Having a fast second dimension makes CE an attractive technique, especially with fast gating methods, which are discussed in Chapter 5. However, these are specialized for specific applications and may require method development techniques specific to CE. 

Fekete S. Rudaz S. Fekete J. Guillaume D. Analysis of recombinant monoclonal antibodies by RPLC Toward a generic method development approach. Journal of Pharmaceutical and Biomedical Analysis, 2012, 70, 158-168. 

There are a number of factors to be considered in method development of RPLC for separation of proteins and peptides. Appropriate pore size is one... 

Column selection during method development often reflects personal preferences or prior experience.1,4,6 Nevertheless, some general guidelines can be suggested based on consensus of experienced chromatographers. Note that these recommendations focus on RPLC. 

This chapter provides an overview of modern HPLC method development and discusses approaches for initial method development (column, detector, and mobile phase selection), method optimization to improve resolution, and emerging method development trends. The focus is on reversed-phase methods for quantitative analysis of small organic molecules since RPLC accounts for 60-80% of these applications. Several case studies on pharmaceutical impurity testing are presented to illustrate the method development process. For a detailed treatment of this subject and examples of other sample types, the reader is referred to the classic book on general HPLC method development by L. Snyder et al.1 and book chapters2,3 on pharmaceutical method development by H. Rasmussen et al. Other resources include computer-based training4 and training courses.5... 

Table 8.4. Summary of Factors Optimized During RPLC Method Development...

I. Jimidar, Lecture presented at the workshop Computer-Assisted RPLC Method Development, Darmstadt,... 

Miller and Hawthorne [416] have developed a chromatographic method that allows subcritical (hot/liquid) water to be used as a mobile phase for packed-column RPLC with solute detection by FID, UV or F also PHWE-LC-GC-FTD couplings are used. Before LC elution the extract is dried in a solid-phase trap to remove the water. In analogy to SFE-SFC, on-line coupled superheated water extraction-superheated water chromatography (SWE-SWC) has been proposed [417]. On-line sample extraction, clean-up and fractionation increases sensitivity, avoids contamination and minimises sources of error. 

Applications Chromatography is a preferred technique for additive analysis as it allows both separation of additives in a mixture and subsequent quantitation. Despite the developments in GC, this technique cannot separate many polymer additives. Even with its lower efficiency in comparison to GC, HPLC is today one of the cornerstones in a polymer additive laboratory. Judging by the number of publications in recent years, HPLC is first among analytical methods for additives (confirmation/identification/quantification). Most additives may be analysed by HPLC if they can be dissolved in an HPLC solvent and absorb UV light. Typical polymer/additive analyses are carried out using LPE followed by HPLC with UV or RI detection [605-611]. Verification of the identity of an analyte is then based on a combination of retention time, UV and RI evidence. RPLC is used most frequently for polymer/additive analysis, but normal-phase and SEC are also used. Consequently, techniques for additive analysis by HPLC are legion. 

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