Quantitative yields of underivatized peptides by HPLC

New methods for peptide separation are being developed. Resolution of peptide mixtures on HPLC with good reproducibility indicates the progress that has already been made in development of supports with useful separation capabilities. At present adsorptive characteristics of the stationary phases may place limitations on the applicability of the technique to underivatized peptides. Hopefully, the application of ionpairing techniques and the development of new support materials will expand the range of application for protein and peptide purification.     

Review: Sample preparation of catecholamines and biologically active peptides by solid phase extraction

This paper reviews solid phase extraction as applied to the sample preparation of catecholamines and biologically active peptides. The mechanisms used in solidphase extraction are: non-polar, polar, ion-exchange, and covalent.     

Separation,detection and quantitation of peptides by liquid chromatography and capillary electrochromatography

This review discusses different liquid chromatographic and capillary electrochromatographic approaches to the separation and quantitation of peptides using silica-based and polymeric-based columns with emphasis on liquid chromatography. Mass spectrometry detection and quantitation of peptides using labeled and label-free procedures, will also be discussed, as well as the effect of amino acids’ properties on the solubility of peptides, an important parameter that influences the selection of the mobile phase. A discussion of different column packing materials, reversed-phase, cyclodextrins, macrocyclic antibiotics, porous graphitic carbon, mixed-phases, and normal-phase will be included, as well as a short discussion of multi-dimensional approaches for the separation of complex peptide mixtures.     

Separation of enantiomers by high performance liquid chromatography using chiral eluents

Simple procedures are presented for separating the enantiomers of α-methyldopa, 5-hydroxytryptophan, tryptophan, triiodothyronine and thyroxine, which require neither special sorbents nor difficult-to-obtain or unstable reagents. The method for α-methyldopa, 5-hydroxytryptophan and tryptophan is based on the use of L-phenylalanine copper complex as the chiral constituent of the mobile phase; LiChrosorb® RP-18 serves as the stationary phase. The procedure for triiodothyronine and thyroxine is grounded on the L-proline copper complex as the chiral reagent and LiChrosorb® Si 60 as the stationary phase. In all observed cases, the D-enantiomer is eluted prior to the respective L-enantiomer. Chirality inversion of the mobile phase (application of the D-phenylalanine copper complex) reverses the order of elution; a racemic eluent (DL-phenylalanine copper complex) leads to no separation. In addition to the enantiomers of α-amino acids, the enantiomers of α-hydroxy acids (mandelic acid) can be separated.     

Part per billion determination of oxygen containing impurities in ammonia-nitrogen mixtures

Ammonia-nitrogen feedstock streams, free of oxygen containing impurities, are essential reagents for fabricating low oxygen contaminated nitride films. An analytical method is described for determining part per billion levels of oxygen containing impurities, which are converted to nitric oxide (NO) through reaction with microwave discharge produced active nitrogen. Quantitative determinations are then based on detecting the resulting chemiluminescence of NO at characteristic wavelengths. This metastable transfer spectrometric method (MTES) provides direct measurements of oxygen impurities over the 14 to 500 ppb region and has dynamic range extending to 200 ppm. The method, unparalleled in its measurement sensitivity for oxygen impurities in flowing nitrogen gas streams, is calibrated with an NBS standard and applied to the determination of the purification efficiency of an on-line resin system for removal of O₂, CO and NO from ammonia-nitrogen mixtures. Gaseous streams, doped deliberately in the 0.2 to 100 ppm level with either O₂, CO or NO following purification were found to contain less than 10 ppb of the residual impurity. A commercially available 1% ammonia in nitrogen semiconductor grade reagent (99.999%) is shown by MTES to be contaminated with 470 ppb of oxygen impurities.     

Separation of (3S, 3′S)-, (3R 3′R)- and (3S, 3′R)-astaxanthin via (−)-camphanic acid esters

A method is described for the qualitative and quantitative determination of configurational isomers of astaxanthin. It is based on the esterification of astaxanthin with (—)-camphanic acid chloride and analysis of the corresponding diesters by HPLC.     

HPLC of fatty acid esters of mono- and polyhydric alcohols. Part 1: Analytical separation

This paper shows how simply and yet very rapidly fatty acid esters of monohydric alcohols, but particularly partial and full fatty acid esters of fully hydric alcohols can be separated and determined by means of high-pressure liquid chromatography on LiChrosorb RP-8 with methanol/water. We have separated quantitatively the methylesters of the fatty acids C_8:0 to C_22:0 and C_24:0, the i-propyl-, i-butyl-, n-hexyl- and i-octyl-esters of the even-numbered fatty acids C_8:0 to C_18:0, mono- and difatty acid esters of the 1,3-bis-(2-hydroxyethyl)-5,5-dimethyl-hydantoin, mono-, di- and triesters of the trimethylolpropane as well as the tetraesters of the penta-erythrite.     

Separation of Amadori peptides from their unmodified analogs by ion-pairing RP-HPLC with heptafluorobutyric acid as ion-pair reagent

Glycation is a common class of nonenzymatic posttranslational modifications relevant for several diseases and cell aging in general, such as D-glucose-derived modifications at the ɛ-amino groups of lysine residues in blood proteins, especially albumin, immunoglobulin, and hemoglobin, for diabetic patients. These Amadori compounds are identified on the peptide level after enzymatic digestion and chromatographic separation by mass spectrometry. Their syntheses usually rely on a global glycation approach. Both areas require the reliable separation of glycated peptides from their unmodified congeners present in different ratios, which is typically not achieved by standard eluent systems in ion-pairing RP-HPLC (IP-RPLC). Here, we compare aqueous acetonitrile and methanol gradients containing either trifluoroacetic acid (TFA) or heptafluorobutyric acid (HFBA) as ion-pairing agents to separate such peptide pairs. TFA-containing eluents resulted in rather low resolutions, and the glycated and unglycated peptides often coeluted. HFBA increased the retention times of the unmodified peptide more than for the glycated peptide thereby improving the separation of all eight studied peptide pairs, even achieving baseline separations for some sequences. Thus the use of HFBA as ion-pair reagent provides a universally applicable eluent system in IP-RPLC to separate glycated peptides from their unmodified counterparts, even at the preparative scale required for synthetic peptides.     

Separation of (3R, 3′ R)-, (3R, 3′ S; meso)-, (3S, 3′ S)- Zeaxanthin, (3R, 3′ R, 6′ R)-, (3R, 3′ S, 6′ S)- and (3S, 3′ S, 6′ S)-Lutein via the dicarbamates of (S)-(+)-α-(1-naphthyl) ethyl isocyanate

A method is described for the qualitative and quantitative determination of configurational isomers of zeaxanthin (=3,3′ -dihydroxy-β, β -carotene) and lutein (=3,3′ -dihydroxy-α -cartotene). It is based on the reaction of these zeaxathin and lutein isomers with (S)-(+)-α-(1-naphthyl) ethyl isocyanate to afford diastereomeric dicarbamates, which are analyzed by HPLC.     

Fluorine-induced chemiluminescence detection of biologically methylated tellurium,selenium, and sulfur compounds

Summary The sensitive detection of volatile, methylated selenium and tellurium compounds based on capillary gas chromatography coupled to fluorine-induced chemiluminescence detection is described. The method requires no sample derivatization, and the detection limits for selenides and tellurides (low pg range) are the lowest reported to date. This technique can resolve and speciate complex mixtures of methylated tellurium, selenium, and sulfur gases and is useful for analysis of selenium and tellurium gases in environmental samples that also contain usually interfering reduced sulfur gases. Applications of the technique for analysis of bacterial and fungal headspace samples are presented.     

Simultaneous use of fluorescence,ultraviolet, and electrochemical detectors in high performance liquid chromatography-separation and identification of phenolic antioxidants and related compounds

Isocratic conditions are described for the separation, identification and quantitation of selected phenolic antioxidants and related compounds by high performance liquid chromatography on a reverse phase microparticulate ODS column. Mobile phases containing 0.05 M lithium perchlorate in aqueous methanol are used. Three detection systems: ultraviolet; fluorescence; and electrochemical detectors are connected in tandem. The use of these three detection systems in sequence as additional means for separation, identification, and quantitation is demonstrated. Other advantages of the system include speed of analyses and improved detection. The ? values and detector responses at 2 settings per detector for 12 compounds are reported.     

Application of Imidazolylazo Resin : Separation of Palladium(II), Silver(I) from Synthetic Mixtures,Medicinal and Geological Samples

ABSTRACT

A resin (I) containing imidazolylazo functional group has been used for the separation and preconcentration of palladium(II) and silver(I) from other metal ions in synthetic mixture, ores, alloys and medicinal samples. No interference could be observed from alkali and alkaline earth metal. Palladium(II) is quantitatively separated from ores and platinum wire without interferences.