Purification by RP-HPLC

The peptide was synthesized on Rink resin (0.2 mmol) using Pfp activated ester of the Fmoc-protected amino acids. All couplings were complete after 2 h. After acetylation, the peptide was cleaved by a 2 min treatment with a 50% TFA soln in CH2C12. After filtration, the TFA soln was concentrated by a stream of N2 to 1-2 mL and the crude peptide was precipitated by adding Et20 (25 mL) and cooling to —78 °C. The Et20 layer was removed and the peptide dried under vacuum. After purification by RP-HPLC pure peptide 53 was obtained yield 101 mg (36%) HRFAB-MS mlz 1408.55180 [M + H]+. 

To the fully derivatized product on the solid support was added excess TFA/CH2CI2 (90 10) and the cleavage reaction was allowed to proceed for 0.5 h at rt. The cleavage solution was then removed by filtration through a cannula and the resin was rinsed with an appropriate solvent (e.g., MeOH/ CH2CI2). Concentration of the combined filtrates provided the crude product 12 in yields > 80% (56-67% isolated yields after purification by RP-HPLC). 

Multidimensional LC SEC fractionation followed by anion-exchange LC and further purification by RP-HPLC. The ICP-MS detector was coupled on-line and ESI-MS and ESI-MS-MS analyses were additionally carried out for identification of the eluted compounds... 

A series of reviews has been published on the analysis of oligonucleotides using CE [183-185]. A comprehensive review on separation of mono, oligo- and polymeric nucleic acids by this method has also been published [186], In contrast to RP-HPLC and ion-exchange chromatography, CE - which has been established as the third technique of choice -is a purely analytical method [187]. Ion-exchange HPLC fails particularly with SODNs [185]. RP-HPLC does not at present allow baseline separation of SODNs, which differ by only one base pair [188]. Thus, control of preparative purifications by RP-HPLC has become an important application of CGE. Table 11 illustrates the resolution efficiency of CGE when compared with other methods. 

The dried product 9 (40 mg) was dissolved in TFA to a concentration not exceeding 20mg-mL 1 and left for 45 min at 20 °C. The majority of the TFA was then evaporated in a stream of air and drying was completed by lyophilization. If 9 had not been purified by RP-HPLC as described above, then purification of 10 was equally possible on the semipreparative column, using a gradient of 25-40% B over 15 min yield 18 mg (32%) ESI-MS mlz [M+H]+ calcd, 609.6 found, 609.6. The FITC derivatives normally elute as doublets on low-loading analytical HPLC because of the use of the commercial mixture of fluorescein 5- and 6-isothiocyanate for the coupling to 8. 

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. 

As shown in the purification of rat histone H2A1 53 amide synthesized using the FMOC strategy, remarkable results using a Pharmacia Biotech Mono S column were obtained for the purification of complex synthetic mixtures (Figures 3 and 4)J5H] This system has been used with similar success with several other peptides. With a complex mixture of peptide/proteins, ion-exchange chromatography was found to be an important first step for preliminary fractionation to be further purified by RP-HPLC. 

Cleavage from the Resin The peptide was simultaneously deprotected and cleaved from the resin by a 30-min treatment with neat TFA (sulfur-containing scavengers have to be absent). The resin was filtered and the soln added to cold MTBE. The precipitated peptide was collected and treated with aq NH3 (pH 9.0-9.5) for 3 h at rt in order to restore the chemical integrity of the nitroxide moiety.1 201 HPLC Purification The peptide was purified by RP-HPLC (MeCN/H20 containing 0.1 % TFA). The above-noted NH3 treatment was repeated immediately after the HPLC elution. 

To improve the purification by adsorption TLC and to reduce the separation of positional isomers by RP-HPLC, Bandi and Ansari (51) reduced the polarity of the hydroxyl groups forming ferf-butyldimethylsiloxy derivatives of PNB esters of monohydroxy fatty acids (PNB-TBDMS-OHFA) (with terr-butyldimethylimidazole in dimethylformamide). 

As an example, two 35-residue peptides were used to form a 70-residue disulfide-bridged AB hetero-stranded molecule. A soln of 1 mg/mL peptide A-SH in EtOH was incubated with DTDP (5 equiv) at rt for ca. 30 min. EtOH was then removed under reduced pressure and the residue was redissolved in 0.05% aq TFA. Excess DTDP and pyridinethiol (HS-TP) were extracted (3x) with equal volumes of Et20 and, depending on the desired purity, the aqueous derivatized peptide (A-S-TP) soln can either be used directly or after purification. A soln of 1 mg-mL 1 peptide B-SH in 0.05% aq TFA (1 equiv) was very slowly added to the A-S-TP soln with an equal volume of degassed 50 mM phosphate buffer containing 1 mM EDTA at pH 7. Both the derivatization and coupling reactions were monitored by RP-HPLC and the product identity was confirmed by ES-MS. 

Final yield. The final yield is the amount of pure product obtained. It is determined by the synthetic yield factors above and also on the recovery during purification on RP-HPLC. If the product cannot be identified in the crude prod-... 

The folding process results in a compact structure with a proportion of the nonpolar side chains buried. This means that, relative to the unfolded form, the folded form has a more polar surface accessible area. This difference is apparent by RP-HPLC, and thus the extent of folding can be readily evaluated by RP-HPLC. The folded material characteristically elutes 1-3 min earlier than the unfolded material. Later eluting peaks contain material that either cannot fold, or did not fold. Since folding separates the material that readily folds from the various byproducts that cannot fold, the folding process actually assists in the purification of the final chemokine product. 

For most chemokines RP-HPLC is the only purification technique needed. However use of only one purification method may not always result in a pure product. In some cases another separation method such as ion-exchange HPLC is needed to obtain purity. This usually resolves contaminants that were not resolved by RP-HPLC. 

A home-made preparative SEC Sephadex G-75 column (30 x 1.9 cm) used for the purification of MT fractions and, after desalting, further separation accomplished by RP-HPLC-UV, ICP-MS and ES-MS detection... 

Chromatographic methods, such as reverse phase HPLC (RP-HPLC) and size exclusion chromatography, were used for identity and purity control, and also for quantitative measurement. The major impurity, which can be detected by RP-HPLC after the two first purification steps (Fig. 3), was isolated and analysed by mass spectrometiy. The mass measured by Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF) mass spectrometry for this impurity was around twice the mass of the monomer (78265 Da), and was interpreted to be a dimer of BBG2Na. 

FSCE can also show impurities present in solid-phase synthesis products that are not clearly evident by conventional purification methods. RP-HPLC with ultra violet (UV) detection is traditionally used to analyze solid-phase synthesis products such as peptoids and peptides as well as to separate them preparatively away from any deletion products. Since each synthetic monomer addition cycle is less than 100% efficientj products that are one or two monomer units shorter than the desired product must be purified out to ensure monodispersity of the final polypeptide or polypeptoid. The separating power of FSCE was compared with analytical RP-HPLC traces after RP-HPLC purification (Figure 12.7). The one peak in the RP-HPLC trace actually separates into one large peak... 

A CPE peptide can be easily prepared by the Fmoc SPPS procedure (Scheme 19). In order to prevent DKP formatiOTi during peptide chain elongation, the dipeptide, Fmoc-Xaa-Cys(Trt)-OH (60), must be introduced onto the Pro-OCHaNH-resin. Standard Fmoc SPPS is then applied to peptide chain elongation, and the usual TFA treatment, followed by RP-HPLC purification, gives the CPE peptide. 

MBHA, and (Met)Expansin (60) resins with an amide linkage. The model tripeptide aldehyde Boc-Phe-Val-Ala-H was synthesized on the three resins. These peptides were checked by RP-HPLC and studied by NMR in CDCI3 without purification. The results showed that peptide aldehydes with high purity could be obtained using this strategy with no detectable trace of racemization (within the limit of sensitivity of NMR). 

Guggjsberg, D., Risse, M., C., and Hadom, R., 2012. Determination of vitamin Bi2 in meat products by RP-HPLC after enrichment and purification on an immunoaffinity column. Meat Science. 90 279 283. 

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