HPLC analyses

Reverse-phase HPLC is the standard analytical tool used for the control of the purity of synthetic peptides. Analysis is most commonly performed on reversed-phase HPLC columns, consisting of 3-5 pm, 100-300 A pore size C8 or C18 silica the larger pore material is generally favoured for the analysis of larger peptides ( 30 residues). Samples are eluted with a gradient formed between water and acetonitrile. An ion-pairing reagent is added to both solvents to improve resolution and selectivity. The most popular buffer systems are listed in Table 3. 

When preparing samples for analysis, it is essential that the peptide is 

Liquid chromatography-mass spectrometry (LC-MS) is an extremely power tool for the analysis of peptides, providing not only information on the purity of the product but also coMormation of structures. The s)rstem typically consists of a microbore HPLC system coupled to an electrospray mass spectrometer. Using such a system the composition of a crude peptide mixture can be quickly determined and by-products identified, enabling synthetic protocols to be rapidly optimized. 

Further, detailed information on HPLC and other techniques that are applied for the analysis of synthetic peptides has been reviewed exten-sively(55,56). 

Besides the basic apparatus for microdialysis perfusions, fraction collection, and HPLC analysis, several additional instruments and devices are needed, depending on where the microdialysis probe is to be implanted. The most complicated instrumental setup is probably that required for brain dialysis. A stereotaxtic instrument and a stereomicroscope are necessary for precise positioning of microdialysis cannulae into various brain structures. Inhalation anesthesia is preferable and more convenient than injections. However, this type of anesthesia calls for additional equipment, such as air lines, valves, and mixing chamber for halothane or other anesthetic gases, as well as good ventilation of the operation theater. 

Conjugated linoleic acid (CLA) has attracted much interest recently, due its potential anti-cancer, anti-atherogenic and anti-inflammatory therapeutic properties (Weiss et al., 2004 see Chapter 3). CLA occurs as several positional and geometric isomers of Ci8 2 with a conjugated double bond. The cis-9, tram-11 CLA isomer occurs most often naturally. Commercial sources of CLA also contain the trans-10 and cis-12 isomer, which is also biologically active. Since various isomers of CLA differ in biological activity, methods of analysis for the various CLA isomers have become very 

Wood (1985) suggested that chloroform might be a more effective extractant. However, chloroform has the disadvantage of forming hydrochloric acid upon storage, and hence its use will lead to the production of phaeophytins and phaeophorbides during the extraction procedure. While this may not seriously affect total analyses, it will surely be noted in HPLC analysis. Thus, if chloroform is to be employed as the extractant, frequent solvent clean-up becomes necessary. 

However, no detailed investigation on the effects of different solvent systems has so far been carried out employing HPLC techniques. For most marine samples extraction with 90 % acetone appears to give satisfactory results. 

In methanol, allomerization of chlorophyll to the corresponding 10-hydroxy- and 10-methoxylactone compounds has been reported to occur instantaneously Schaber et al., 1984). This is the main reason why methanol should be avoided as the extraction agent. Lyo-philisation of filtered material results in pigment losses Riaux-Gobin et al., 1987). 

Storage of natural seston at low temperatures (-20 °C) for up to five months has no effect on seston samples stored diatom culture material, however, showed considerable compositional changes after only one month. This has been attributed to ongoing chlorophyllase activity. Jeffrey and Hallegreef (1987) also reported on potential problems associated with enzymatic chlorophyll degradation. Thus, samples rich in phytoplankton should be extracted as soon as possible after collection the extracts can then be stored deep-frozen for up to five months with only minor changes. 

Extraction should be carried out in subdued light at low temperatures. If cell disintegration is deemed necessary, either ultrasonication or cell grinding (manual or automated) may be used. In our experience, 3 min of ultrasonication followed by low temperature centrifugation (4°C) for 30 min at 6000 rpm is sufficient to extract well over 90% of the pigments in particulate material and to remove particulate debris. Hie supernatant may be analysed by HPLC without any further clean-up. In fact, additional handling should be avoided as some carotenoids are extremely sensitive to oxidation. 

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The enantiomeric excess of 3.10c has been determined by HPLC analysis using a Daicel Chiracel OD column and eluting with a 60 / 1 (v/v) hexane(HPLC-grade) / 2-propanol(p.a.) mixture. At a flow of 1 ml per minute the rentention times for the different isomers of 3.10c were 6.3 min. (exo, major enantiomer) 7.1 min. (exo, minor enantiomer) 7.7 min. (endo, major enantiomer) 10.7 min. (endo, minor enantiomer). 

Description of Method. Fluoxetine, whose structure is shown in Figure 12.31a, is another name for the antidepressant drug Prozac. The determination of fluoxetine and its metabolite norfluoxetine. Figure 12.31 b, in serum is an important part of monitoring its therapeutic use. The analysis is complicated by the complex matrix of serum samples. A solid-phase extraction followed by an HPLC analysis using a fluorescence detector provides the necessary selectivity and detection limits. 

One advantage of an HPLC analysis is that a loop injector often eliminates the need for an internal standard. Why is an internal standard used in this analysis What assumption(s) must we make about the internal standard ... 

A iridine traces in aqueous solution can be determined by reaction with 4-(p-nitroben25l)pyridine [1083-48-3] and potassium carbonate [584-08-7]. Quantitative determination is carried out by photometric measurement of the absorption of the blue dye formed (367,368). Alkylating reagents interfere in the determination. A iridine traces in the air can be detected discontinuously by absorption in Folin s reagent (l,2-naphthoquinone-4-sulfonate) [2066-93-5] (369,370) with subsequent chloroform extraction and hplc analysis of the red dye formed (371,372). The detection limit is ca 0.1 ppm. Nitrogen-specific thermal ionisation detectors can be used for continuous monitoring of the ambient air. 

There are four main theaflavins common to black teas, and a second group of minor theaflavins, including the isotheaflavins (55) and neotheaflavins (57) (Table 5). The total theaflavin concentration in black tea leaves does not usually exceed 2% and can be as low as 0.3%. At most, only 10% of the catechins in tea flush can be accounted for as theaflavins in black tea and the fate of the remaining catechins is less clear. Theaflavins can be readily determined by direct hplc analysis of tea beverages (48,58,59). 

Coimnonly used methods for tlie measurement of pestieides in water samples involve. Solid Phase Exttaetion (SPE) followed by Gas Chromatography or High Performanee Liquid Cliromatography (HPLC) analysis. SPE is a multiple step proeedure and, thus, time eonsuming. 

APPLICATION METHANOL ELUENT RP-HPLC ANALYSIS FOR ESTERS OF 4-AMINOBENZENTHIOSULFINIC ACID AT PROCESS OF THEIR SYNTHESIS... 

A powerful tool now employed is that of diode array detection (DAD). This function allows peaks detected by UV to be scanned, and provides a spectral profile for each suspected microcystin. Microcystins have characteristic absorption profiles in the wavelength range 200-300 nm, and these can be used as an indication of identity without the concomitant use of purified microcystin standards for all variants. A HPLC-DAD analytical method has also been devised for measurement of intracellular and extracellular microcystins in water samples containing cyanobacteria. This method involves filtration of the cyanobacteria from the water sample. The cyanobacterial cells present on the filter are extracted with methanol and analysed by HPLC. The filtered water is subjected to solid-phase clean-up using C g cartridges, before elution with methanol and then HPLC analysis. 

HPLC analysis of anatoxin-a was first carried out by Astrachan and Archer, " who extracted the toxin from Anabaenaflos-aquae using chloroform followed by hydrochloric acid. The HPLC analysis was carried out on an ODS column using hypochlorate-methanol. Other systems used since include acetic acid extraction and analysis on a reversed-phase C g column using methanol-water mobile phase, and extraction in water after ultrasonication and analysis on reversed-phase... 

W.S. Hancock, J.T. Sparrow, HPLC Analysis of Biological Compounds A Laboratory Guide, (Chromatographic Science Series, Vol 26), Marcel Dekker, New York, 1984. ISBN 0824771400. 

The second example is the SE-HPLC analysis of recombinant hGH. In this example, SE-HPLC is used for both a purity and a protein concentration method for bulk and formulated finished products. This method selectively separates both low molecular weight excipient materials and high molecular weight dimer and aggregate forms of hGH from monomeric hGH, as shown... 

Absolute configurations of the isoxazolidines obtained in the nitrone cydoaddition reactions described in Schemes 7.21 and 7.22 were determined to be 3S,41 ,5S structure by comparison of the optical rotations as well as retention times in a chiral HPLC analysis with those of the authentic samples. Selection of the si face at C/ position of 3-crotonoyl-2-oxazolidinone in nitrone cydoadditions was the same as that observed in the Diels-Alder reactions of cyclopentadiene with 3-croto-noyl-2-oxazolidinone in the presence of the J ,J -DBF0X/Ph-Ni(C104)2-3H20 complex (Scheme 7.7), and this indicates that the s-cis conformation of the dipolaro-phile has participated in the reaction. 

Gel permeation ehromatography (GPC)/normal-phase HPLC was used by Brown-Thomas et al. (35) to determine fat-soluble vitamins in standard referenee material (SRM) samples of a fortified eoeonut oil (SRM 1563) and a eod liver oil (SRM 1588). The on-line GPC/normal-phase proeedure eliminated the long and laborious extraetion proeedure of isolating vitamins from the oil matrix. In faet, the GPC step permits the elimination of the lipid materials prior to the HPLC analysis. The HPLC eolumns used for the vitamin determinations were a 10 p.m polystyrene/divinylbenzene gel eolumn and a semipreparative aminoeyano eolumn, with hexane, methylene ehloride and methyl tert-butyl ether being employed as solvent. 

High-pressure pumps operating at up to 6000 psi are required to force solvent through a tightly packed HPLC column, and electronic detectors are used to monitor the appearance of material eluting from the column. Alternatively, the column can be interfaced to a mass spectrometer to determine the mass spectrum of every substance as it elutes. Figure 12.18 shows the results of HPLC analysis of a mixture of 10 fat-soluble vitamins on 5 jam silica spheres with acetonitrile as solvent. 

Figure 12.18 Results of an HPLC analysis of a mixture often fat-soluble vitamins.

Yields estimated by HPLC analysis. b bp 77 -87 C/0.5 Torr. c Numbering refers to the product. 

C and the residual solid was dried in vacuo over P205 overnight and then heated in toluene (20 mL) under reflux until TLC showed that the reaction was complete (15 min). The sodium p-toluenesulfinate produced was filtered off and the filtrate was washed with H20 (2 x 50 mL), dried and evaporated to leave the product yield 0.40 g (78%) oil bp 80 C/10 Torr. HPLC analysis (silica column at 0 T7) showed that the tautomers 7a and 8a were present in the ratio 5.9 1. 

Fig. 4.1.6 HPLC analysis of a sample of purified natural aequorin on a TSK DEAE-5PW column (0.75 x 7.5 cm) eluted with 10 mM MOPS, pH 7.1, containing 2mM EDTA and sodium acetate. The concentration of sodium acetate was increased linearly from 0.25 M to 0.34 M in 14 min after the injection of the sample. Full-scale 0.02 A. Flow rate 1 ml/min. Reproduced with permission, from Shimomura, 1986a. the Biochemical Society.

To a solution of 516 nig (1 mmol) of the stannane 17 (R = C.H3, 95% ee) in 10 ml of dry DME is added, at — 78 C, 0.62 nlL of 1.6 M BuLi (1 mmol) in hexane. After 5 min gaseous carbon dioxide is bubbled into ihe yellow solution. The reaction mixture decolorizes immediately. After a further 10 min the reaction is quenched with sat. aq NH4C1. The organic layer is extracted with 2N aq NaOll. Acidification of the aqueous layer with 2N aq IICI is followed by extraction with F.t,o, drying over Na,SC)4 and concentration in vacuo yield 193 mg (92%) 95% ec [determined by HPLC-analysis of the amide obtained by derivati/alion with (S)-x-phenylelhylaminc] [a]20 — 37 (r = 1,6, CI1C1,). 

In a flame-dried Schlenk tube 0.37 g(1.88 mmol) of (-)-3-exo-(dimethylamino)isoborneol (C) and 200 mL of dry toluene are placed under an atmosphere of argon. 27 mL of 4.2 M diethylzinc (113 mmol) in toluene are added and the resulting solution is stirred at 15°C for 15 min. After cooling to — 78°C, lOg (94.2 mmol) of benzaldehyde are added and the mixture is wanned to O C. After stirring for 6 h, the reaction is quenched by the addition of sat. NH4C1 soln. Extractive workup is followed by distillation yield 12.4 g (97%) 98% ee [determined by HPLC analysis. Baseline separation of rac-1 -phenyl-1 -propanol was achieved on a Bakerbond dinitrobenzoyl phenylglycine column (eluent 2-propanol/hexanc 1 3 flow rate l.OmL/ min detection UV 254 nm)] [a] 0 —47 (c = 6.11, CHC13). 

Sl-a-Phenylethylaminc (2a 94% ee) and (.S)-2-heptylamine (2b 82% ec) were synthesized from enantiomerically pure nitrones 1 a and 1 b (33-39% overall yield). The enantiomeric purity of the amines was determined by HPLC analysis of their 3,5-dinitrobenzamidines on a D-naphthyl alanine column,... 

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