Parameters affecting high performance liquid chromatographic separations of neurohypophyseal peptide hormones (original) (raw)
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Journal of Chromatography A, 2009
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.
Journal of Chromatography A, 1983
Weak anion-exchange and reversed-phase high-performance liquid chromatographic methods for peptide separations were compared using a tryptic digest of "rat small myelin basic protein". In these experiments, a number of tryptic peptides that were-not resolved on the reversed-phase column could be separated on the weak anion-exchange column, and in other instances, as might be expected, reversed-phase chromatography provided better resolution of certain peptides than did the weak anion-exchange method. The results obtained strongly suggest that the combined use of these two methods of separation, which utilize different selectivities, can provide an excellent improvement in resolving power for a number of peptide separations.
Journal of Chromatography A, 2007
Chromatographic measurements were made on 17 physicochemically diversified high-performance liquid chromatography (HPLC) columns which were further analyzed in terms of their similarities and dissimilarities for 25 carefully designed, structurally diverse peptides showing distinctly distinguished groups. The goal of the study was to investigate the molecular mechanism of retention and to find an objective manner of quantitative comparison of retention properties and classification of modern stationary phases for reverse phase HPLC (RP-HPLC). We utilized the structural descriptors of peptides obtained from molecular modeling to describe their chromatographic retention behavior under given HPLC conditions. Quantitative structure-retention relationships (QSRR) with the following descriptors were employed: logarithm of the sum of amino acid retention contributions in a given peptide, log Sum AA , logarithm of the Van der Waals volume of the peptide, log VDW vol , and logarithm of its calculated n-octanol/water coefficient, clog P. The best QSRR equations were obtained in the case of monolithic and regular octadecylsilica columns. On the other hand, the combination of QSRR and principal component analysis (PCA) can be considered as the efficient tool allowing column classification and searching for orthogonal HPLC conditions required to separate peptides.
Stationary phases for peptide analysis by high performance liquid chromatography: a review
Analytica Chimica Acta, 1997
A survey is given of modern stationary phases employed in high performance liquid chromatography (HPLC) analysis of peptides. The physico-chemical properties of peptides and their consequences for the selection and optimization of the separation system are briefly discussed, followed by a summary of the approaches to the selection and characterization of stationary phases. The properties and applicability of various stationary phases are then critically reviewed, including aspects such as size-exclusion, ion-exchange, reversed-phase, hydrophobic-interaction, affinity and chiral systems, as well as some specialized separation techniques. Emphasis is placed on the most recent literature. 0 1997 Elsevier Science B.V.
Neuropeptides, 1991
A reverse-phase, high-performance liquid chromatographic (HPLC) method was employed to separate and characterise five neuropeptides from complex mixtures, with important advantages over methods employed earlier using combined HPLC-RIA studies. Peptides were separated using 0.5M pyridine-0.5M formic acid buffer, pH 4, containing propan-l-01 14% (met-enkephalin, leu-enkephalin, neurotensin) or 20% (CCK-S-S, substance P) at a flow rate of l.Oml/min. Isocratic conditions, and volatile solvents, resulted in a highly reproducible method, producing samples in a form designed for subsequent RIA. The application and importance of the procedure is demonstrated by comparison of the measurements of apparent peptide levels in crude brain extracts with those of authentic peptides as determined after HPLC purification.
Journal of Chromatography A, 1980
A high-performance liquid chromatographic system employing a chemically bonded alhyl phase and a tetraalkylammonium phosphate bufber has been evaluated for separation of synthetic mixtures of hypothalamic oligopeptides. Separation of two peptides in the mixture having very simiiar adjusted retention times has been achieved. Sensitivity down to 5 ng is attained. Peptides range in sire from three to 31 amino acids. Field desorption mass spectra illustrate novel structural elucidation methods for individual peptides and signal a quantitkation method. I~ODUCIION This paper describes a high-petiormance liquid chromatogmphic (HPLC) method to separate mixtures of biological oligopeptides in a fast and facile manner, optimizing sensitivity, speed and resolution of the separation to provide a purified peptide fraction for quantification and structural elucidation studies. Towatds this end, the triethylamine phosphate (TEAP)-acetonitrile system is used with a @3ondapak C,, HPLC column. Synthetic mixtmes of hypothalamic oligopeptides coutaining from three to 31 amino acids are employed_ A field desorption (FD) spectmm of an underivatized hcxapeptide illustrates the utility of the novel ionization method in peptide chemistry to provide molecular ions of underivatized oligopeptides and for quantifkation. Reversed-phase (RP) HPLC using chemically bonded al&l stationary phases has become an important and powerful tool in peptide separation chemistry. Long chain al@1 silanes are chemically bonded to silanol groups located on the silicon surface of the packing material. An end-capping reaction follows where trimethylsiiyl groups are chemically bonded to most of the re maining unreacted silanol groups. Various authors reviewed HPLC column technology and provided a theoretical basis for separations afforded by RP col~unns~-~. HPLC retention indices related to compound lipophilicity were estimated'. Ho&&h and co-worker&' show separation on RP columns is a fbn&on of increasin g hydrophobic&y of the peptide-paired-ionsolvent hydration complex.. ml-9673p~ @0225
Optimisation of high performance liquid chromatography separation of neuroprotective peptides
Journal of Chromatography A, 2005
The study of experimental design conjunction with artificial neural networks for optimisation of isocratic ion-pair reverse phase HPLC separation of neuroprotective peptides is reported. Different types of experimental designs (full-factorial, fractional) were studied as suitable input and output data sources for ANN training and examined on mixtures of humanin derivatives. The independent input variables were: composition of mobile phase, including its pH, and column temperature. In case of a simple mixture of two peptides, the retention time of the most retentive component and resolution were used as the dependent variables (outputs). In case of a complex mixture with unknown number of components, number of peaks, sum of resolutions and retention time of ultimate peak were considered as output variables. Fractional factorial experimental design has been proved to produce sufficient input data for ANN approximation and thus further allowed decreasing the number of experiments necessary for optimisation. After the optimal separation conditions were found, fractions with peptides were collected and their analysis using off-line matrix assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF-MS) was performed.
Journal of Chromatography B: Biomedical Sciences and Applications, 1996
HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta-and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C~8 stationary phase and mobile phases of 20-40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4-5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50 mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the D-and L-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM fl-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.
Chromatographia, 2004
A method for determination of some biologically active penta-and nona-peptides under isocratic conditions in capillary liquid chromatography was developed. Separation system consisting of XTerra C18 stationary phase and mobile phase composed of a mixture of acetonitrile with 0.1% trifluoroacetic acid (TFA) and water with 0.1% TFA in the ratios 75/25 (v/v) and 85/ 15 (v/v) was suitable not only for a good resolution of enkephalin and vasopressin related peptides, respectively, but it also enabled separation of the respective biopeptides from other constituents of human urine. Calibration curves for the studied peptides were linear in the measured concentration range from 1.00 to 1.57·10 )2 mg mL )1 . The limit of detection and limit of quantification were in the range of units of lg mL )1 and tens of lg mL )1 , respectively; slightly higher values were obtained for nonapeptides. Determination of certain biologically active peptides in urine can serve in future as a tool for diagnosis of various diseases, e.g. autism.