Prep HPLC

After cleovoge from the resin the crude peptide is dissolved in oqueous ocetic acid and purified via prep. HPLC. 

Application of Prep-HPLC to PGS Analysis. Reverse phase liquid chromatography has proven to be well suited for cleanup of plant extracts by prep-HPLC (4, 46, 47, 48). When the mobile phase is initially an aqueous buffer at pH 2.8, all but the highly charged (e.g., zeatin ribotide with 5 AMP used as a representative compound for zeatin ribotide) plant hormones are retained at the head of the column (Fig. 1). Since the PGS are retained, samples can be injected onto the column in a dilute form. In-... 

Fractions are collected on the basis of the retention times of the respective PGS standards. The remaining portions of the column effluent are diverted to waste. Figure 3 diagramatically represents the sequence of events used for prep-HPLC of PGS samples. 

Automation of Prep-HPLC. Reverse phase prep-HPLC separation has proven to be a very reproducible technique. For this reason, the process can be automated. In addition to the standard components of an HPLC system, the following are required for automation an autoinjector capable of large volume injections (2.0 to 5.0 ml), a programmable controller (a microprocessor controller), a fraction collector, and a waste valve (3-way valve) controlled by a solenoid (Fig. A). The microprocessor should allow programming of the solvent flow rate, the sequence for solvent gradient formation, the time of injection, advancement of the fraction collector (to collect specific fractions rather than just uniformly incremented advancement), and control of a waste valve. 

The use of GLC-EC has become a well accepted method for the analysis of ABA as described by Saunders (37). The purity of ABA (methyl ester) detected on this system may be confirmed by forming the trans-ABA isomer methyl ester in sunlight while in acetone and rerunning the sample. We have found that prep-HPLC is useful in the purification of plant extracts for ABA analysis by GLC-ED (45). Another unique identification method takes advantage of the extreme cotton effect that ABA exhibits. The degree of optical rotation can be used for quantification of ABA if the sample is highly purified (37). 

The capability of analyzing a complex mixture in a chromatographic run by the hyphenation of several techniques, such as NMR and MS, to HPLC is becoming more popular in the pharmaceutical industry. NMR and MS data on the same analyte are crucial for structural elucidation. When different isolates such as metabolites are analyzed by NMR and MS, one cannot always be certain that the NMR and the MS data apply to the same analyte, especially when the analytes have been isolated using analytical columns and prep columns for the MS and NMR analysis, respectively. HPLC conditions are not always reproducible when analytical and prep-HPLC columns are used to isolate different amounts of the analytes of interest. To avoid this ambiguity, LC-MS and LC-NMR are combined. MS data should be obtained initially because with NMR, data collection in the stop-flow mode can take hours or days, depending on the complexity of the structure and the amount of sample. This is why it is preferable to designate this operation as LC-MS-NMR rather than LC-NMR-MS or LC-NMR/MS. 

Cmde pCCK-33 was dissolved in the mixture of acetonitrile and 0.05 M (pH = 6.5) ammonium acetate buffer and was purified by prep-HPLC on a Cl8 column. The synthetic peptide was identified by using the native pCCK-33 standard. Pure fractions were pooled and lyophilized three times. 

Chapter 10 details the use of column chromatography for the isolation of impurities. It also discusses the various options that are available for stationary phases and analytical detectors, as well as the current equipment available for this work. The choice of purification techniques (prep HPLC, low-pressure silica columns, etc.) is also discussed. Finally, a procedure is described for both analytical methods development as well as scale-up to preparative columns. 

M. Verzele and C. Dewaele, Prep HPLC A Practical Guideline, TEC, 1986. 

Figure 10.2. Mass spectro- and UV traces before and after purification by prep-HPLC.

Figure 10.3. Purity distributions of a pyrrolopyrrole library A) before and (B) after purification by prep-HPLC. This figure is a summary of high-throughput parallel LCVMS results. LC/MS was carried out using a MUX-LCT LC/MS system with eight parallel channels.

Co-eluting impurity. Our prep HPLC method was of lower resolution than that of analytical. Impurities eluting closely to the target compound may get collected in the same fraction. 

The purity of both monomers for the Stille polymerization is crncial. This will not only ensure an accurate stoichiometric control (see Section 15.2.4 for details), but also minimize possible negative impact to the device properties by these impurities. Typically, both distaimylated donor monomer and dibromo acceptor monomer need to be recrystallized or purified by prep HPLC prior to the polymerization, in order to ensure a high level of purity. An excellent case study demonstrating monomer purity s effect on the molecular weight of the D-A copolymer was provided by Osaka et al. In this study, the authors used three different procedures to purify both monomers and observed essentially no difference in the NMR spectra of these monomers after the different purification processes. However, the purity of the monomers significantly affected the observed molecular weight of their polymers (from 13 kg/mol to 73 kg/mol), which noticeably influenced the photovoltaic properties of devices based on these polymers. 

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