Normal-phase HPLC, purification

F-HPLC to give 17 crude individual products 70-syn/anti and 71. These crude products were not isomerically pure. Removal of the fluorous tag and hydantoin formation was achieved by treatment of the individual amides 70-syn/anti and 71 with diisopropylethyl-amine (DIPEA) under microwave conditions. The cyclative cleavage reactions of 10-syn and 10-anti provided the same products 72. Normal-phase HPLC purification gave 11 of 12 possible final products 72 and 73. 

Id (Scheme 3) . This was prepared via the corresponding phosphite, which can be synthesized by reaction of the alcohol with chlorodiethyl phosphite and triethylamine. The phosphite then undergoes nucleophihc substitution reaction with anhydrous H2O2 forming the hydroperoxide Id (enantiomeric ratio S/R 65/35) and isomeric hydroperoxide le in a 2 1 mixture in 74% overall yield starting from the alcohol. Purification was possible by normal-phase HPLC. So in this case transformation of the phosphite to the hydroperoxide proceeds with partially retained configuration. 

Thus, in order to evaluate the enantiopurity of the Michael product 14a obtained during the optimization of the catalyst A (Scheme 5), the purification of the analytical quantities of the crude 14a from its diastereomer and residual p-ketoester 12a was performed by semipreparative normal phase HPLC using achiral silica-based column ( -hexanes/lPA, Zorbax Rx-SIL column). This purification provided partial separation, and the pure fractions containing major diastereomer (i.e., 14a) along with some mixed fractions containing both diastereomers were collected. Remarkably, the first collected fraction contained highly enantiopure 14a (99% ee). 

Chromatography. A number of HPLC and TLC methods have been developed for separation and isolation of the brevetoxins. HPLC methods use both C18 reversed-phase and normal-phase silica gel columns (8, 14, 15). Gradient or iso-cratic elutions are employed and detection usually relies upon ultraviolet (UV) absorption in the 208-215-nm range. Both brevetoxin backbone structures possess a UV absorption maximum at 208 nm, corresponding to the enal moeity (16,17). In addition, the PbTx-1 backbone has an absorption shoulder at 215 nm corresponding to the 7-lactone structure. While UV detection is generally sufficient for isolation and purification, it is not sensitive (>1 ppm) enough to detect trace levels of toxins or metabolites. Excellent separations are achieved by silica gel TLC (14, 15, 18-20). Sensitivity (>1 ppm) remains a problem, but flexibility and ease of use continue to make TLC a popular technique. 

For the final purification, a sequence of normal phase chromatography, size exclusion chromatography, reversed-phase (RP)-HPLC and other techniques are used. There are no general rules as to how to proceed but, due to the high capacity and low irreversible absorption of Sephadex, size exclusion should be used in the very beginning, whilst HPLC is better employed for the final purification steps. 

The decision about which HPLC column to choose is really controlled by the separation you are trying to make and how much material you are trying to separate and/or recover. I did a rather informal survey of the literature and my customers 15 years ago to see which columns they used. I found 80% of all separations were done on some type of reverse-phase column (80% of those were done on C18), 10% were size separation runs (most of these on polymers and proteins), 8% were ion-exchange separations, and 2% were normal-phase separation on silica and other unmodified media, such as zirconium and alumina. The percentage of size- and ion-exchange separations has increased recently because of the importance of protein purification in pro-teomics laboratories and the growing use in industry of ion exchange on pressure-resistant polymeric and zirconium supports. 

A reverse-phase HPLC assay, as part of the Association of Official Analytical Chemists report on analysis of fat-soluble vitamins, was described by DeVries et. al. (65). Analysis were made with a Merck LiChrosorb RP-18 column (Manufacturing Chemists, Inc., Cincinnati, OH) and a acetonitrile propionitrile water (79 15 6) mobile phase. Although adequate chromatography was realized, the authors were concerned that problems arose concerning influence of temperature, dissolution of sample and purification of solvents in the mobile phase. For these reasons they recommended normal-phase chromatography. Separation of vitamins D2 and Dj with their systems was not discussed. 

Separation and purification of three turmerones, e.g. ar-turmerone, a- and P-turmerone, from turmeric oil extracted by supercritical carbon dioxide gave 71% purify by weight. Subsequently, purification using a normal-phase silica gel 60 column could separate and purify three major turmerones with 86% purify by weight of ar-turmerone and 81% purity by weight of a- and P-turmerone. These were identified by liquid-solid chromatography, NMR qualification and HPLC quantification, respectively (Li-Hsun Chang et al., 2006). 

NP-HPLC Normal-phase liquid chromatographic methods applying Diol-columns or common silica columns are well suited for the analysis of the total steryl ferulate content. They require very little sample preparation, as total lipid extracts can frequently be directly injected into the column without purification or fractionation. Run times for SFs are also relatively short, and a good separation from other lipid components can be obtained in less than 10 min in traditional HPLC systems. Depending on the column type and the sample, SFs elute as one or two peaks. Two peaks are obtained from the separation of SFs, which have ferulic acid both in cis- and trans- configuration (Nystrom et ah, 2008). The relative retention time (obtained with a silica column and hexane/ethyl acetate 97 3 as eluent) of the cis- form is about 0.5 smaller than that of steryl irans -ferulates (Akihisa et al., 2000). 

Lewis acid-mediated ene reaction of di- —)- / ,25)-2-phenyl-1-cyclohexyl diazenedicarboxylate with cyclohexene using tin(IV) chloride in dichloromethane at —60 °C for 5 min afforded the azo-ene adduct in 80% yield after purification by flash chromatography (eq 2). The H NMR spectrum of the azo-ene adduct recorded at 380 K in deuterated toluene established the presence of only one diastereomer. Further analysis of the ene adduct by HPLC on a Whatman Partisil 5 normal phase silica column using hexane-ethyl acetate (9 1) as eluent confirmed the presence of only one diastereomer. 

Interest in phosphorus-containing calixarenes continues. Structures reported include hexa(diethoxyphosphoryloxy)calix[6]arene (8), inherently chiral 1,2-bridged calix[4]arene diphosphates, and a calixarene like C3 symmetric receptor with a phosphate function at the cavity bottom. " The purification of phosphate substituted calixarenes has been studied by chiral HPLC and by normal reverse phase HPLC. Mono(6-0-diphenoxyphosphoryl)-P-cyclodextrin (9) and mono(6-0-ethoxyhydroxyphosphoryl)-p-cyclodextrin (10) have been synthesised and show enantioselective inclusion of D and L amino acids e.g. 3.6 for D/L serine in the case of 9). ... 

A UV-triggered purification system was described by Kibby [44] in support of the purification of combinatorial libraries generated at Parke-Davis. This system is operated in either reverse-phase or normal-phase mode, and is employed as well for chiral separations. Multiple column sizes allow the system to accommodate the purification of samples in weight up to 50 mg. The operational protocol involves an initial scouting run by analytical HPLC with APCI-MS detector. The conditions that are selected are based on structural information. Fraction collection is controlled by customized software, and sample identity, UV, MS data along with chromatographic data are imported from the analytical LC-MS. Peaks are collected only when the UV threshold is met within an appropriate collection window thus, the number of fractions obtained is limited. Postpurification loop injection mass spectra are collected on these fractions to determine the desired component from each sample. 

Since peptides for research purposes are usually required in only mg to g amounts, the time-saving solid-phase peptide synthesis method148-501 can be used. The strategy is in principle similar to that in solution, with the difference that there is no need for isolation of the intermediate products. As the growing peptide chain is synthesized on a suitable resin the whole procedure lends itself to automation. The drawback is that every reaction step at the resin has to be forced to give an almost 100% yield. In practice, this cannot be accomplished, with the consequence that the desired product must be isolated from a mixture of side-products by the final, normally HPLC, purification procedure which is sometimes difficult to perform and also expensive. Peptides of up to 50 amino acid residues are now readily accessible using stepwise solid-phase procedures[501. 

Normal-phase LC is a less potent alternative [132,133] and has been recommended only when impurities in the crude were derived from the loss of side-chain protecting groups. If the crude product exhibits a significant amount of deletion peptides, then purification by reversed-phase HPLC techniques is required [131]. In this case, Cg or even C4 columns are the most suitable for peptides of —10 residues. For longer peptides the less polar diphenyl-based phase is recommended. 

---