Reverse phase method development examples

Are there any other analytical protocols available from the analytical chemist The more information about a compound, the easier it will be to develop a purification procedure. For example, in addition to the reversed-phase method, are there thin-layer chromatography (TLC) methods available for this compound This could help in deciding whether a silica column may be the appropriate tool for this separation. 

The selection and development of a reversed-phase method can be a straightforward process, and for this reason, many methods have been developed on reversed-phase SPE, especially the C-18 sorbent due to its reliable nature. The following examples describe the reversed-phase extraction of compounds of differing polarities from various matrices, as an indication of the broad extent to which reversed-phase SPE can be applied. 

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... 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

An example of a simple CZE method for peptide analysis and characterization is the one developed for protegrin IB-367.37 IB-367 is a peptide containing 17 amino acid residues that possess antimicrobial properties, and it is being developed for treatment of oral mucositis associated with aggressive cancer chemotherapy as well as other topical applications. This polycationic product was chemically synthesized using solid-phase and purified by preparative reversed-phase HPLC. IB-367 is rich in cysteine and arginine residues. 

LC-NMR can be used to identify natural products in crnde plant extracts that usually consist of complex mixtnres. The crnde natural product extracts normally contain a great nnmber of closely related and difficult-to-separate compounds. The classical separation approach may become very tedious and time-consuming. The directly conpled HPLC-NMR presents an efficient separation techniqne together with a powerfnl spectroscopic method to speed up the identification process. LC-NMR has been nsed extensively for characterization of natnral prodncts. More recently, the combination of LC-NMR and LC-MS has been further developed in this field. Eor example, Wilson et al. have nsed combined on-flow NMR and electrospray ionization MS to characterize ecdysteroids in extracts of silene otites. After reversed-phase HPLC nsing D2O in acetonitrile-dj and UV detection, the LC flow was split 95 5 for the simnl-taneous detection by NMR and MS. The peaks of interest were analyzed by stop-flow NMR to give better quality spectra for structural assignment. 

The choice of the proper stationary and mobile phases for the foregoing purpose would depend on several factors, such as the nature (polarity, stability in mobile phase) of the NOC analyzed and the availability/compatibility of the detector used. For example, if only a TEA is available as a detector, the use of an ion-exchange or a reversed-phase system is ruled out, because both require aqueous mobile phase for proper operation. Moisture in the mobile phase causes freeze-up of the cold traps in the TEA and also results in noisy response due to interference during chemiluminescence detection. Similarly, if one is using, as the detector the newly developed Hi-catalyzed denitrosation-TEA (62) or the photolytic cleavage-TEA (58), a reversed-phase system using aqueous mobile phase would be the method of choice. These detectors, however, have not been demonstrated to work in the normal-phase system. The use of an electrochemical detector will also be incompatible with an organic solvent as the mobile phase. 

Successful separations can be carried out only by planning and careful experimentation, the details of which are discussed extensively in Chapter 5. In one sense there are too many ways to achieve a separation in LC. But while this makes the first choice of where to start difficult, the good news is that there are many ways to achieve success. By looking at Figure 4-1 it is obvious that the use of the reverse-phase mode in LC has broad applicability and is, in fact, the most used mode of LC. Reverse phase is used for 80-85% of the separation problems encountered by users of HPLC. For this reason, the majority of Chapter 5, on developing methods, is devoted to reverse-phase examples. Additionally, Chapter 11 is a useful experiment to experience the method development aspect of this mode. Chapter 9 is a useful experiment to experience method development in the normal phase mode. 

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