Reversed-phase HPLC materials

Recovery and Purification. The dalbaheptides are present in both the fermentation broth and the mycelial mass, from which they can be extracted with acetone or methanol, or by raising the pH of the harvested material, eg, to a pH of 10.5—11 for A47934 (16) (44) and A41030 (41) and actaplanin (Table 2) (28). A detailed review on the isolation of dalbaheptides has been written (14). Recovery from aqueous solution is made by ion pair (avoparcin) or butanol (teicoplanin) extraction. The described isolation schemes use ion-exchange matrices such as Dowex and Amberlite IR, acidic alumina, cross-linked polymeric adsorbents such as Diaion HP and Amberlite XAD, cation-exchange dextran gel (Sephadex), and polyamides in various sequences. Reverse-phase hplc, ion-exchange, or affinity resins may be used for further purification (14,89). 

Amino acid analysis, by reverse-phase HPLC, of acid-hydrolyzed uncross-linked recombinant resilin and cross-linked recombinant resilin clearly shows the presence of dityrosine in the cross-linked sample (Figure 9.3c). Further evidence of the presence of dityrosine was obtained by UV irradiation (Xmax,ex 315 nm Xmax,em 409 nm). Dityrosine endows natural resilin with pH-dependent blue fluorescence [38] on UV irradiation. The cross-linked recombinant resilin material was similarly fluorescent, strongly suggesting dityrosine cross-links. 

The stationary phase matrices used in classic column chromatography are spongy materials whose compress-ibihty hmits flow of the mobile phase. High-pressure liquid chromatography (HPLC) employs incompressible silica or alumina microbeads as the stationary phase and pressures of up to a few thousand psi. Incompressible matrices permit both high flow rates and enhanced resolution. HPLC can resolve complex mixtures of Upids or peptides whose properties differ only slightly. Reversed-phase HPLC exploits a hydrophobic stationary phase of... 

In comparison to GC, an advantage in using HPLC is that there is no decomposition of the acidic forms of cannabinoids. Commonly reversed-phased (RP) materials are used as the stationary phase. Mostly the octadecyl-type... 

Acetochlor and its metabolites are extracted from plant and animal materials with aqueous acetonitrile. After filtration and evaporation of the solvent, the extracted residue is hydrolyzed with base, and the hydrolysis products, EMA and HEMA (Figure 1), are steam distilled into dilute acid. The distillate is adjusted to a basic pH, and EMA and HEMA are extracted with dichloromethane. EMA and HEMA are partitioned into aqueous-methanolic HCl solution. Following separation from dichloromethane, additional methanol is added, and HEMA is converted to methylated HEMA (MEMA) over 12 h. The pH of the sample solution is adjusted to the range of the HPLC mobile phase, and EMA and MEMA are separated by reversed phase HPLC and quantitated using electrochemical detection. 

A disadvantage of the reversed-phase HPLC technique or that of Derenbach et al. [82] is that it is only semi-quantitative. Against this is the fact that the technique using silanised porous glass is easy to keep free of contamination, has a comparatively high adsorption capacity, and permits the fractionated desorption of accumulated material. 

As an example, Table 4.3.1 presents the results calculated from reversed-phase and normal-phase HPLC analysis, respectively, of samples taken from an industrial WWTP [16]. Concentrations were expressed in microgram per gram of material as received. In the water sample, reversed-phase HPLC analysis showed the presence of NP and NPEO (Fig. 4.3.7(A)) with levels of 0.008 and 0.383 p,gg 1 (sum of NPEO), respectively. Normal-phase analysis of NPEO oligomers... 

The actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom two-step method that employs chromatography to separate the Amb a 1 from the other extracted proteins. The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of purified Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purification steps, and the Bradford assay adequately reflects Amb a 1 concentration through the remainder of the process. 

Figure 2 (right). Reverse-phase HPLC elution profile of the tumor-localizing fraction of HPD isolated by non-aqueous gel exclusion chromatography. (B) Hydrolysis of this material in 50% aqueous THF containing IM HCl, at 37 °C for 24 hours. (C) After hydrolysis in IM NaOH, 50% aqueous THF under the same condition. The major porphyrins resulted are hematoporphyrin (HP), hydroxyvinyl deutero-porphyrin (HVD), and protoporphyrin (PP). 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

FW Quackenbush. Reverse phase HPLC separation of cis- and trans-carotenoids and its application to/3-carotenes in food materials. J Liq Chromat 10 643-653, 1987. 

There are a large number of other published procedures for the separation of a number of sweeteners and preservatives at one time these are all based on reverse-phase HPLC. Perhaps one of the most startling is the method published by Williams (1986). This uses a small particle size (3 xm) C8 column and allows the separation of a range of colours, sweeteners and preservatives in less than 5 min. The materials separated were amaranth, quinoline yellow, quinine sulphate, sunset yellow, caffeine, aspartame, saccharin, vanillin, sorbic acid, benzoic acid and green S. 

We were also able to use FAB mass spectrometry to determine the amino acid sequence around the active site serine in the acyl transference domain of rabbit mammary fatty acid synthase.6 The synthase was labelled in the acyl transferase domain(s) by the formation of O-ester intermediates after incubation with [" " C]-acetyl- or malonyl-CoA (Fig. 2A). The modified protein was then digested with elastase (Fig. 2B), and radioactive material isolated via successive purification steps on Sephadex G-50 and reverse phase HPLC. The isolated peptides were then sequenced by FAB MS. The data summarized in Table II established the sequences of both the acetyl and malonyl hexapeptides to be N-acyl-Ser-leu-Gly-Glu-Val-Ala. 

While HPLC grade water is commercially available, I have found it to be expensive and to have limited shelf life. The best technique for purifying water seems to be to pass it through a bed of either reverse-phase packing material or of activated charcoal, as in a Milli-Q system. Even triple distillation tends to co-distill volatile impurities unless done using a fractionation apparatus. 

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