Preparative HPLC

Note Every analytical separation can be performed in preparative scale  

Practical High-Performance Liquid Chromatography, Fourth edition Veronika R. Meyer 2004 John Wiley Sons, Ltd ISBN 0-470-09377-3 (Hardback) 0-470-09378-1 (Paperback) 

This chapter describes the method for separating amounts varying from 10 mg up to several grams, there being no essential differences for variations above or below these limits. 

Guiochon, A. Felinger, D.G. Shiraz and A.M. Katti, Fundamentals of Preparative and Nonlinear Chromatography, Elsevier Academic Press, New York, 2nd edn, 2006 H. Schmidt-Traub, ed.. Preparative Chromatography, Wiley-VCH, Weinheim, 2005 G. Guiochon, J. Chromatogr. A, 965, 129 (2002). 

Practical High-Performance Liquid Chromatography, Fifth edition Veronika R. Meyer 

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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). 

Phosphate Esters. The phosphorylation of sucrose using sodium metaphosphate has been reported (78). Lyoptulization of a sodium metaphosphate solution of sucrose at pH 5 for 20 hours followed by storage at 80°C for five days produced a mixture of sucrose monophosphates. These products were isolated by preparative hplc, with a calculated yield of 27% based on all organic phosphate as sucrose monoesters. Small proportions of glucose and fmctose were also formed. 

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.

Fig. 3-9. Preparative HPLC of 100 mg of the test racemate 8 in a single 2 mL injection using a 250 x 4.6 mm i.d. column containing (5)-Glu-(5)-Leu-DNB CSP. Conditions mobile phase ethyl acetate, flowrate 2.0 mL min , UV detection at 380 nm. Injection 2 mL of 50 mg mL racemate solution. Fractions collected before and after the indicated cut point were 98.4 % ee and 97 % ee pure, respectively. (Reprinted with permission from ref. [86]. Copyright 1999, American Chemical Society.)...

Samples of PB-DPE were subjected to RP-HPLC producing the chromatographic patterns shown in Figure 1. The two major peaks of Penta (Fig. lA, peaks 1 and 2), were isolated by preparative HPLC and analyzed by NMR. The spectra (Table 1) were in excellent agreement with previously published data (refs. 3-6). The two HPLC peaks were identified as 2,2, 4,4 tetrabromo DPF and 2,2, 4,4, 5 pentabromo DPF, respectively. 

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). 

Sequence information for the remaining fragments was obtained by Edman degradation (see Section 5.3.1 above) after isolation of the individual peptides using preparative HPLC - the chromatographic resolution being sufficient to allow this, and thus enabled the complete sequence to be determined. 

Phengl-2(lH)Pyridone-Propylsulfonate Sodium Salt Vllb. A solution of 3-phenyl-2(lH)pyridone sodium salt (0.264 g, 1.25 mmol) and 3-bromopropylsulfonate sodium salt (0.241 g, 1.25 mmol) in acetonitrile (650 mL) was brought to reflux with stirring for 24 h. The yield was 41%, as determined by HPLC. An analytical sample was obtained from the filtered residue of reaction by preparative HPLC. Anal. Calcd for Ci4Hi4NS04Na H2O C, 50.45 H, 4.8 N, 4.2. Found C, 50.38 ... 

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. 

Thus, racemic acid 12 (R = H) was obtained by [3+2] cycloaddition in 90-95% yield (Scheme 5.9) [28]. Its resolution into enantiomers could be achieved either by chiral preparative HPLC, or by fractional crystallization of its cinchonidine salts. Better results were obtained upon enzymatic kinetic resolution of its iso-butyl ester 12 (R = i-Bu) [29]. However, further work showed that racemic thiolester 13, which... 

In 1996, Fu et al. reported the S3mthesis of the planar chiral heterocycles 64, formally DMAP fused with a ferrocene core [82]. While the original synthesis provided racemic 64a in only 2% overall yield requiring a subsequent resolution by preparative HPLC on a chiral stationary phase, a recently improved synthesis furnished the racemic complexes 64 in 32-40% yield over seven steps. A subsequent resolution with di-p-toluoyltartaric or dibenzoyltartaric acid gave access to the enantiomers with >99% ee (28 14% yield for each isomer in this step) [83]. 

Prior to semi-preparative HPLC, matrix compounds may be present that require a purification step. In some cases, high sugar or salt contents and also pectic-like substances may preclude concentration and isolation precipitation using 2-propanol or ethanol is then required. Subsequent filtration will yield a pigment solution and a colorless filtrate that may be rinsed with a mixture of one part water and two parts alcohol for complete discoloration. The filtrate volume will be reduced under vacuum before further purification. Usually, precipitation precedes desalting. 

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. "... 

Hotchkiss, Jr., A. T., Haines, R. M., and Hicks, K. B., Improved gram-quantity isolation of malto-oligosaccharides by preparative HPLC, Curb. Res., 242, 1, 1993. 

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). 

Preparative HPLC of component II was difficult. Component II was collected with a resolution comparable to that shown in Figure 2. Higher resolution showed the presence of at least 5 minor peaks. Slowing the water/methanol gradient finally produced a single peak, which when collected, totaled 14 mg of slightly yellow oil from 2.1 gms OFAs extracted with ODS silica (16). Structural work on II has not yet appeared in the literature, so more detail is included. 

Thermal degradation of Irganox 1076 in air was studied by means of HPLC-UV/VIS and by preparative HPLC-NMR. At 180 °C cinnamate and dimeric oxidation products are formed, and at 250 °C de-alkylation products are observed [660], On-line LC-NMR hardly covers a real need in polymer/additive analysis, as the off-line option is mostly perfectly adequate for that purpose. 

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-... 

Many small proteins, in particular those that function extracellularly (e.g. insulin, GH and various cytokines) are quite stable and may be fractionated on a variety of HPLC columns without significant denaturation or decrease in bioactivity. Preparative HPLC is used in industrial-scale purification of insulin and of IL2. In contrast, many larger proteins (e.g. blood factor VIII) are relatively labile, and loss of activity due to protein denaturation may be observed upon high-pressure fractionation. 

The major disadvantages associated with HPLC include cost and, to a lesser extent, capacity. Thus, for both technical and economic reasons, preparative HPLC is employed almost exclusively... 

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