Reversed-phase chromatography method development

Fig 13. Final polishing step of recombinant epidermal growth factor, using reversed phase chromatography. Method developed on pre-packed RESOURCE RPC and scaled up on... 

The actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom two-step method that employs chromatography to separate the Amb a 1 from the other extracted proteins. The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of purified Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purification steps, and the Bradford assay adequately reflects Amb a 1 concentration through the remainder of the process. 

Foda et al. have developed a reversed-phase chromatography method using C18 reversed-phase analytical column. 

Purify the biotinylated protein or molecule using dialysis or gel filtration. For small molecule biotinylation where these separation methods may not be appropriate, other procedures may have to be developed, such as reverse-phase chromatography or organic precipitation techniques. 

The three isomers of cresol are not as readily separated by HPLC, although recent techniques have been developed to accomplish this task. Reversed-phase chromatography columns have been used for the analysis of cresols with limited success. Recently, a new reversed-phase support has been developed that allows complete separation of the three cresol isomers (Bassler and Hartwick 1989). Inclusion complexes of the cresols with p-cyclodextrin cleanly separate the three isomers on commercially available columns (Yoshikawa et al. 1986). Detection limits down to 1 ppm can be obtained by this method. 

With all these tools, you can usually find a way to separate the components of a mixture if it does not contain too many compounds. If reversed-phase chromatography fails, normal-phase chromatography or one of the methods in Chapter 26 could be appropriate. Method development is part science, part art, and part luck. 

Based on the fact that aromatic sulfonic and carboxylic acids were successfully separated by reversed-phase chromatography in the presence of organic electrolytes, Chaytor and Heal (158) developed a method for the separation of 15 synthetic colors using a mobile phase containing o-phosphoric acid (Table 7). The presence of the electrolyte provided lower variation in response and retention over a period of time. Furthermore, eluted peaks were sharper than those seen in ion-pair chromatography. 

RP HPLC is an area of intensive research. Over 5000 papers are published yearly on the theory, development, and practical apphcations of reversed-phase chromatography. In this chapter we present our vision of the current state of the RP HPLC. We hope that it will be useful for practical chro-matographers in their efforts to develop efficient and selective separation methods, and we also hope that it will be encouraging for researchers studying different aspects of HPLC separations. 

The first step in method development is selecting an adequate HPLC mode for the particular sample. This choice depends on the character of the sample compounds, which can be either neutral (hydrophilic or lipophilic) or ionic, low-molecular (up to 2000 Da) or macromolecular (biopolymers or synthetic polymers). Many neutral compounds can be separated either by reversed-phase or by normal-phase chromatography, but a reversed-phase system without ionic additives to the aqueous-organic mobile phase is usually the best first choice. Strongly lipophilic samples often can be separated either by non-aqueous reversed-pha.se chromatography or by normal-phase chromatography. Positional isomers are usually better separated by normal-phase than by reversed-phase chromatography and the separation of optical isomers (enantiomers) requires either special chiral columns or addition of a chiral selector to the mobile phase. 

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