Separation preparative HPLC

More recendy the cis and trans isomers of the mosquito repellent CIC-4, a mixture of citroneUa isomers, have been separated by preparative hplc and bioassayed for effectiveness (23). Chiral-phase capillary gas chromatography and mosquito repellent activity of some oxazoUdine derivatives of (+)-and ( —)-citroneUal have been studied to find stmcture—activity relationships (24). Several 2-aLkyl- -acetyloxahdines have been synthesized and tested against mosquitoes, with further efforts using nmr to determine the rotational isomers of the more active N-acetyl-2,2-dimethyloxazohdine (25). 

Enantiomeric separations have become increasingly important, especially in the pharmaceutical and agricultural industries as optical isomers often possess different biological properties. The analysis and preparation of a pure enantiomer usually involves its resolution from the antipode. Among all the chiral separation techniques, HPLC has proven to be the most convenient, reproducible and widely applicable method. Most of the HPLC methods employ a chiral selector as the chiral stationary phase (CSP). 

Table 3-1. Values of enantiomeric resolution of DNP-amino acids in a running electrolyte containing the three fractions 1, 2, and 3 of the cyclo(Arg-Lys-X-Pro-X-(3 Ala) sublibrary separated by preparative HPLC.

Of the five remaining peaks (Figure IB, peaks 5 to 9), three (peaks 5,7 and 9), were easily separated from the octa samples by preparative HPLC, and the two remaining (peaks 6 and 8) were isolated from high-melting (Figure 1C). 

Although these Boc derivatives underwent methylation with poor selectivity (compared to 3-amino-N-benzoyl butanoates [106] and Z-protected methyl 4-phen-yl-3-aminobutanoate [107]), epimers were succesfully separated by preparative HPLC or by flash chromatography. However, saponification of the methyl ester caused partial epimerization of the a-stereocenter and a two-step (epimerization free) procedure involving titanate-mediated transesterification to the corresponding benzyl esters and hydrogenation was used instead to recover the required Boc-y9 -amino acids in enantiomerically pure form [104, 105]. N-Boc-protected amino acids 19 and 20 for incorporation into water-soluble /9-peptides were pre-... 

We further synthesized unsymmetrical MiniPHOS derivatives 13b (Scheme 13) [30]. Thus, enantioselective deprotonation of l-adamantyl(dimethyl)phos-phine-borane (74, R = 1 -Ad), followed by treatment with ferf-butyldichlorophos-phine or 1-adamantyldichlorophosphine, methylmagnesium bromide and bo-rane-THF complex afforded the optically active diphosphine-boranes 82b as a mixture with the corresponding raeso-diastereomer. Enantiomerically pure unsymmetrical MiniPHOS-boranes 82b were obtained by column chromatography on silica gel or separation by recycling preparative HPLC. 

Forthright isolation of standard substances from known sources may be achieved by analytical and/or semi-preparative HPLC. Although it appears promising with respect to obtainable pigment yields, countercurrent chromatography has been applied only once for red beets but lacked sufficient separation efficiency. "... 

Resolution of the enantiomers of anti-BPDE was achieved by reaction of the racemic dihydrodiol with (-)-menthoxyacetyl chloride followed by preparative HPLC separation and basic methanolysis to give the optically pure (+) and (-) dihydrodiols (18-20). Epo-... 

A similar strategy was also applicable for the synthesis of six- and eight-membered siloxanol-ring systems. Hydrolysis of z-PrRSiCl2 (R = Ph, o-Tol) with ZnO and KOH provided the six-membered siloxane rings as a mixture of two constitutional isomers cis-trans-(i-PrRSiO)3 (539, R = Ph 540, R = o-Tol) and Wzr-(z-PrRSiO)3 (541, R = Ph 542, R = o-Tol) which were separated by preparative HPLC. Subsequent reaction with HC1/A1C13 and hydrolysis of the chloro intermediates yielded the same product for both isomers, namely Wzr-[z-Pr(OH)SiO]3 543, implying that isomerization occurs under these conditions (Scheme 75).484... 

Preparation of 4-12-cvclohexenvloxv )-stvrene. A stirred mixture of 34.36g (0.096 mole) methyltriphenylphosphonium bromide and 10.75g (0.096 mole) potassium t-butoxide in 200ml dry THF is treated drop-wise with a solution of 16.16g (0.080 mole) of 4-(2-cyclohexenyl)-benzaldehyde in 30ml THF under inert atmosphere. Once the addition of aldehyde was completed, the mixture was stirred at room temperature for another 2 hours. Ether and water were then added to the reaction mixture until clearly separated phases were obtained with no solid residue. The organic layer was separated and washed three times with water, dried over magnesium sulfate and evaporated. The resulting semi-solid was triturated in 10% ethyl acetate-hexane mixture to remove most of the triphenylphosphine and the evaporated extract was purified by preparative HPLC using hexane as eluent. This afforded 9.35g (58%) of the pure monomer, which was fully characterized by H and C-NMR as well as mass spectrometry. 

Various liquid chromatographic techniques have been frequently employed for the purification of commercial dyes for theoretical studies or for the exact determination of their toxicity and environmental pollution capacity. Thus, several sulphonated azo dyes were purified by using reversed-phase preparative HPLC. The chemical strctures, colour index names and numbers, and molecular masses of the sulphonated azo dyes included in the experiments are listed in Fig. 3.114. In order to determine the non-sulphonated azo dyes impurities, commercial dye samples were extracted with hexane, chloroform and ethyl acetate. Colourization of the organic phase indicated impurities. TLC carried out on silica and ODS stationary phases was also applied to control impurities. Mobile phases were composed of methanol, chloroform, acetone, ACN, 2-propanol, water and 0.1 M sodium sulphate depending on the type of stationary phase. Two ODS columns were employed for the analytical separation of dyes. The parameters of the columns were 150 X 3.9 mm i.d. particle size 4 /jm and 250 X 4.6 mm i.d. particle size 5 //m. Mobile phases consisted of methanol and 0.05 M aqueous ammonium acetate in various volume ratios. The flow rate was 0.9 ml/min and dyes were detected at 254 nm. Preparative separations were carried out in an ODS column (250 X 21.2 mm i.d.) using a flow rate of 13.5 ml/min. The composition of the mobile phases employed for the analytical and preparative separation of dyes is compiled in Table 3.33. 

Hydroxyacyl analogues were prepared as a mixture of two diastereomers. In the case of 3-hydroxyhexanoyl (R=C3H7), the two diastereoisomers were successfully separated by HPLC. The absolute stereochemistry at the 3-hydroxy centre in either of the isomer was not established [15]. 

Once an assessment on a particular impurity has been made all process-related compounds will be examined to confirm that the impurity of interest is indeed an unknown. An easy way of doing this is to compare the retention times of known process-related compounds to that in question. If this analysis confirms that the compound is an unknown, the next step would be to obtain an LC-MS on the compound. Mass spectrometry provides structural information which aids in determining structure. In some cases, mass spectrometry will be enough to identify the compound. In other cases, more complicated methods like LC-NMR are needed or the impurity will need to be isolated in order to obtain additional information. Compounds that are not purified often contain high levels of by-products and can be used for this purpose. Alternatively, mother liquors from crystallizations also contain levels of by-products. Other ways of obtaining larger quantities of impurities include flash chromatography which is typically used for normal phase separations or preparative HPLC which is more common for reversed phase methods. Once a suitable quantity of the compound in question has been obtained a full characterization can be carried out to identify it. 

Recently, an improved pretreatment of the plant material combined with preparative HPLC separation has been published for the isolation of anhydrovincaleukoblastine (8) from the leaves of C. roseus 27). It has also be shown that the yield of 8 could be enhanced by treatment of acidic aqueous extracts of C. roseus with sodium borohydride, suggesting that... 

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