Preparative HPLC purified

If the specific binding is not saturable or the nonspecific binding is >20%, it is important to determine whether unconjugated fluorochrome is present in the FP-Fl preparation. HPLC-purified FP-Fl should be free of unconjugated fluorochrome. Unconjugated fluorochrome binds to cells and causes nonspecific fluorescence. Repeated freezing and thawing of FP-FL can cause dissociation of the fluorochrome and should be avoided. 

Semi-preparative HPLC" was then used to further purify the extract. Instrumentation and techniques were the same as described previously. Active compounds were contained in the 7-11 min fraction (data not shown). 

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. 

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. 

The 4 mM soln of the crude peptide [H-(Gly-Pro-Hyp)5-Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-Hyp-Gly-Ile-Leu-Gly-Cys(Acm)-Cys-Gly-Gly-OH] 28 in degassed argon-sat. DMF/AcOH (95 5) was added dropwise to a 100 mM soln of di[5-nitro(2-pyridyl)]disulfide (5 equiv) in DMF/AcOH (95 5) with exclusion of air oxygen. The reaction was monitored spectroscopically at 430 nm, and after completion (1 to 2 h), the solvent was removed under reduced pressure. The resulting residue was dissolved in H20 and the excess reagent was filtered off. The H20 was removed under reduced pressure and the residue was reprecipitated from TFE with methyl /ert-butyl ether and purified by preparative HPLC to give 29 yield 13% the product was characterized by MALDI-TOF-MS, HPLC, and amino acid analysis. 

The reduced form of Na+, K+-ATPase inhibitor-I (10) was obtained by treatment of the protected peptide synthesized by the soln procedure with HF, followed by reaction with Hg(OAc)2. After purification of the crude product on Sephadex G-25, the reduced peptide (110 mg) was dissolved in 0.1 M NH4OAc buffer (1L, pH 7.8) at a peptide concentration of 0.018 mM and then stirred at rt. After 24 h, the major peak in the HPLC, which coeluted with the natural product, corresponded to 55% of the product distribution. The mixture was acidified to pH 3 with AcOH and 10 was purified by RP-HPLC. When the oxidation was carried out in the presence redox reagents at a peptide/GSH/GSSG ratio of 1 100 10, after 24 h the major oxidation product increased to 69%. The mixture was acidified with AcOH and the product (10) isolated by preparative HPLC yield 20%. The product was characterized by MALDI-TOF-MS and amino acid analysis a combination of enzymatic peptide mapping and synthetic approaches were applied to assign the cystine connectivities. 

The linear peptide thioester was dissolved in 0.2 M phosphate buffer (pH 7.2, 0.1-0.6 mM soln) containing 50% DMF and TCEP (6 equiv). After completion of the reaction (3-24 h, monitored by HPLC) the cyclic peptide was purified by preparative HPLC (small amounts of byproducts result from hydrolysis of the peptide thioester). 

To Boc-Ala-f/V-(4- 4-[2-(trimethylsilyl)ethoxycarbonyl]butoxy]benzyl)]Ile-OAI (2.0 g, 2.82 mmol) in THF (20 mL) was added under stirring at rt 1 M TBAF in THF (3 mL) dropwise, and saponification of the ester proceeded for 3 h. The H20 (100 mL) and AcOH (3 mL) were added to the mixture. The acid was extracted into EtOAc and the combined EtOAc extracts were washed with brine and H20, and dried (MgS04). The solvent was removed, and the resulting oil was purified by preparative HPLC (C18, 2.2 x 25 cm 0-60% MeCN over 60 min) to give a colorless oil yield 2.54 g (44%) (note this is the yield given in the original paper, clearly it is incorrect). 

HPLC. The crude product was dissolved in 0.1% aq TFA (10 mL), filtered, and purified by preparative HPLC [Preppak, Cartridge YMC ODS column (4.8 x 30 cm), gradient elution MeCN/0.1% TFA/H20 from 20 to 50% MeCN in 90 min (flow rate of 50mL-min-1)]. The fractions containing pure product were combined, concentrated, and lyophilized yield 22.4mg (44%) the peptide was characterized by HPLC (purity 98%), amino acid analysis, and FAB-MS. 

To the monocyclic peptide (120mg, 0.138mmol) in dry DMF (138mL), PyBOP (1 equiv) and DIPEA (3 equiv) were added and the soln stirred at rt overnight. The solvent was removed and the residue lyophilized from MeCN/H20. The crude peptide was purified by preparative HPLC as described above yield 59 mg (50%) the peptide was characterized by FAB-MS. 

An alternative access was achieved by alkylation of the a-diphenylphosphino acetaldehyde SAMP hydrazone 95, yielding the hydrazone products 96 in good yields (60-63%) and good diastereomeric excesses (die = 68-71%) as EjZ mixtures, from which the major diastereomer was separated and purified by preparative HPLC. Ozonolysis and in-situ reduction with the borane-dimethyl sulfide complex of the aldehydes generated gave the air-stable borane-protected 2-diphenylphosphino alcohols 97 in good yields (67-83%). Reaction with DABCO afforded the unprotected 2-phosphino alcohols 98 in very good yields (85-91%) and excellent enantiomeric excesses (ee > 96%) (Scheme 1.1.27). 

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