High-performance liquid chromatography determination of phenolic components in wine using off-line isotachophoretic pretreatment

The off-line combination of isotachophoresis (ITP) and high-performance liquid chromatography (HPLC) to improve sample pretreatment and determination of phenolic compounds in wine was investigated. The ITP system provided an enhanced sample load capacity and served as a sample clean-up technique, HPLC performed a final separation of the analytes presented in samples. The phenolic components were separated by Discovery RP Amid C16 chromatography column using water-methanol-acetonitrile-orthophosphoric acid gradient. The identification of phenolic compounds was made by comparison of the retention data obtained for the standard mixture, pretreated sample and the sample spiked standard additions. Satisfactory recoveries for all components analysed were observed between 86.1 and 109.2%.     

Isolation from fetal bovine serum of a peptide similar to the alpha chain of thrombin by reversed-phase high-performance liquid chromatography

During the preparation of erythrotropin from fetal bovine serum, a group of peptides co-eluted with this erythroid cell stimulating factor on semi-preparative reversed-phase high-performance liquid chromatography. They could be subsequently separated by a combination of reversed-phase high-performance liquid chromatography in the presence of heptafluorobutyric acid as ion-pairing reagent and gel permeation high-performance liquid chromatography. One of these peptides has been extensively purified. Partial amino acid sequence analysis indicated that fourteen of the seventeen N-terminal amino acids are identical with the N-terminal sequence of the alpha chain of bovine thrombin. The same isolation procedure could be useful for the identification of other major peptides of fetal bovine serum.     

Reversed-phase high-performance liquid chromatography: preparative purification of synthetic peptides

Biologically active peptides synthesized by the solid phase methodology of Merrifield were purified by reversed-phase high-performance liquid chromatography using newly developed preparative radially compressed cartridges fitting Waters Assoc . Prep LC 500 liquid chromatograph. Cartridges were handpacked with Vydac C18, C4 or diphenyl derivatized silicas (pore size 300 A) of different particle sizes (10-20 micron). Large scale purification of gram amounts of gonadotropin releasing hormone analogs (agonist and antagonist) as well as amidated human pancreatic tumor growth hormone releasing factor (a 40-peptide) illustrate the resolutive power of this technique applied to the isolation of more than 300 synthetic peptides in our laboratory over the last two years. Difficult separations were achieved by changing supports (C18, C4, diphenyl) as well as mobile phase composition: (triethylammonium phosphate pH 2.25 or 6.5, 0.1% trifluoroacetic acid, ammonium acetate pH 6.5 and acetonitrile). Protected amino acids and peptides amenable to normal-phase chromatography on Vydac spherical underivatized silica were purified economically by the reversed-phase mode. It is understood that this general, convenient and versatile strategy may be applicable to the preparative scale isolation of any other class of compounds usually separated on reversed-phase high-performance liquid chromatography.     

Separation and quantification of angiotensins and some related peptides by high-performance liquid chromatography

A high-performance chromatographic technique for the separation of angiotensins and some related peptides is described. Complete separation of angiotensin I, angiotensin II, tetradecapeptide and the tetrapeptide Leu-Val-Tyr-Ser is achieved in a single step, using reversed-phase high-performance liquid chromatography. The application of this technique for the detection of renin activity in crude biological samples, employing the artificial renin substrate tetradecapeptide, is demonstrated.     

Peptide maps at picomolar levels obtained by reversed-phase high-performance liquid chromatography and pre-column derivatization with phenyl isothiocyanate. Microsequencing of Phenylthiocarbamyl Peptides

A new reversed-phase high-performance liquid chromatography approach to the production of analytical peptide maps by pre-column derivatization using phenylisothiocyanate is described. Tryptic peptide digests were derivatized with phenyl isothiocyanate to form the phenylthiocarbamyl peptides followed by reversed-phase high-performance liquid chromatographic analysis. The phenylthiocarbamyl peptides were separated by reversed-phase high-performance liquid chromatography with the conventional gradient elution system of water-acetonitrile containing trifluoroacetic acid. The sensitivity of detection of these peptide derivatives was within the range 5-10 pmol with a constant baseline at 254-260 nm. The isolated phenylthiocarbamyl peptides can be subjected to automatic Edman degradation. The effectiveness of this method was exemplified by microsequencing of phenylthiocarbamyl peptides isolated from tryptic digests of three different proteins: alpha-lactalbumin, beta-lactoglobulin and a lambda light-chain immunoglobulin.     

Comparison of reversed-phase liquid chromatography and hydrophilic interaction/cation-exchange chromatography for the separation of amphipathic alpha-helical peptides with L- and D-amino acid substitutions in the hydrophilic face

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is a novel high-performance technique which has excellent potential for peptide separations. Separations by HILIX/CEX are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlaid on ionic interactions with the cation-exchange matrix. In the present study, HILIC/CEX has been compared to reversed-phase liquid chromatography (RP-HPLC) for separation of mixtures of diastereomeric amphipathic alpha-helical peptide analogues, where L- and D-amino acid substitutions were made in the centre of the hydrophilic face of the amphipathic alpha-helix. Unlike RP-HPLC, temperature had a substantial effect on HILIC/CEX of the peptides, with a rise in temperature from 25 to 65 degrees C increasing the retention times of the peptides as well as improving resolution. Our results again highlight the potential of HILIC/CEX as a peptide separation mode in its own right as well as an excellent complement to RP-HPLC.     

Recent progress in carbohydrate separation by high-performance liquid chromatography based on hydrophilic interaction

This article surveys recent developments in the separation and analysis of carbohydrates by high-performance liquid chromatography, in adsorption or partition modes, on polar sorbents with less polar eluents, a technique that is now termed hydrophilic interaction chromatography. A variety of chromatographic methods are included under this generic heading, the most important being adsorption chromatography on silica and partition chromatography on silica-based sorbents bearing bonded polar phases. Examples are given of the applications of these stationary phases, as well as the newer polymer-based polar sorbents, in high-performance liquid chromatography of carbohydrates and their derivatives.     

Sensitive method of detection, quantitation and purification of peptides using pre-column derivatization with phenyl isothiocyanate

A sensitive method for the detection, quantitation and purification of peptides is described. The method is based on pre-column derivatization of peptides with phenyl isothiocyanate to form phenylthiocarbamoyl derivatives (PTC peptides). The derivatized peptides are analysed by reversed-phase high-performance liquid chromatography on a Zorbax ODS column (5 micron) and detected at 269 nm with a sensitivity limit of 1-5 pmol. The technique was utilized for the separation of a mixture of closely related synthetic peptides. The eluted PTC peptides were collected with an average recovery yield of 75% as determined by amino acid analysis. This method of separation of PTC peptides was also combined with the determination of the complete structure of recovered PTC-dynorphin A-(1-13) using the solid-phase sequenator (Sequemat). The advantages of the derivatization method are the rapidity and completeness of the reaction, the stability of the product, the sensitivity and specificity of the detection of derivatized peptides and the compatibility of the technique with subsequent analytical procedures. A particular application of this method was exemplified by the dosage of enkephalins secreted from perfused bovine adrenal glands.     

Resolution and quantitation of apolipoproteins A-I and A-II from human high-density lipoprotein by size exclusion high-performance liquid chromatography

Apolipoproteins A-I and A-II, extracted from human high-density lipoprotein (HDL), were resolved and quantified by size exclusion high-performance liquid chromatography on TSK 125 and TSK 250 analytical columns connected in series without the use of chemical denaturants or detergents in the eluent buffer. The columns were pre-equilibrated with a solution containing 0.1 M sodium phosphate, pH 7.2, 0.2 M sodium chloride at a flow-rate of 1 ml/min. Delipidated HDL (1 mg protein per ml) was resolved into two populations of apolipoprotein (apo) A-I: one representing the apo A-I monomer and the other, a self-associated form with a molecular weight of approximately 120,000 daltons. The column eluates were screened for immunoreactivity to apo A peptides, and the identity of each peak was confirmed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis followed by immunoblot analysis. Apo A-I peptides isolated by high-performance liquid chromatography disrupted unilamellar phospholipid vesicles to form smaller phospholipid particles that eluted on gel filtration columns within the size range of HDL. Thus, a rapid method for the isolation and quantitation of non-denatured apolipoproteins from HDL has been developed using size exclusion high-performance liquid chromatography.     

Characterization of an apolipoprotein C-III mutant by high-performance liquid chromatography and time-of-flight secondary ion mass spectrometry

Apolipoprotein (apo) C-III isoforms from a patient with a mutant apo C-III and from controls were isolated to homogeneity by isoelectric focusing and subjected to proteolytic digestion. The peptides obtained were separated by reversed-phase high-performance liquid chromatography, and their molecular masses were determined by time-of-flight secondary ion mass spectrometry. Molecular masses of peptides derived from apo C-III0, C-III1 and C-III2 were indistinguishable from control preparations, whereas the mutant apo C-III contained a COOH-terminal, carbohydrate-containing peptide with an abnormal retention time in high-performance liquid chromatography and a molecular mass higher by 291 daltons owing to oversialation at position 74 of the amino acid sequence (apo C-III3).     

Isotachophoretic separation of selected imidazolium ionic liquids

Results of determination of selected imidazolium ionic liquids by isotachophoresis (ITP) with conductometric detection was presented. The effects of the molar mass of different ionic liquids on electrophoretic mobility was observed. The presented method was validated and basic validation parameters were determined. Limit of detection (LOD) in a 10 and 25 ng/L for anions and cations, respectively, is very satisfied. Thanks to its low cost and high rate, the presented method can be used in qualitative routine analysis as an alternative technique to liquid chromatography.     

Analysis of the aqueous phase of human cervical mucus by reversed-phase high-performance liquid chromatography and capillary isotachophoresis

The aqueous phase of human cervical mucus was analysed by reversed-phase high-performance liquid chromatography (HPLC) and capillary isotachophoresis (ITP). With HPLC, seventeen ultraviolet-absorbing and eight fluorescent components and with ITP five anionic and four cationic components could be determined. The sample pre-treatment consisted of a simple ultrafiltration. Ten samples from fertile women and eleven samples from infertile women were analysed. In six samples from the infertile group higher median concentrations of several components were found. This may be an indication of disturbances in the biochemical processes of the cervical mucus of woman with fertility problems.     

Fluorescence derivatization of bioactive peptides for high-performance liquid chromatography

New fluorescence derivatization techniques are introduced for the quantitative determination of arginine- or tyrosine-containing peptides by high-performance liquid chromatography with fluorescence detection. The methodology offers enhancement of both sensitivity and specificity. It is thus suitable for trace (0.1–10 pmol) analysis of the bioactive peptides such as angiotensins and enkephalins.     

Identification of food-derived bioactive peptides in blood and other biological samples

Recent studies have demonstrated the presence of food-derived peptides in human blood after ingestion of enzymatic hydrolysates of food proteins, while most peptides in food are degraded into amino acids during digestion and absorption. To capture and clarify the food-derived peptides in blood, solid-phase extraction (SPE) using a mini-spin column packed with a strong cation exchanger was developed. This technique allows the use of a nonvolatile acid such as trichloroacetic acid, a strong protein denaturant, for the deproteinizing procedure. To improve resolution of hydrophilic peptide and increase specificity and sensitivity in the detection of peptide by reversed-phase high-performance liquid chromatography (RP-HPLC) after subfractionation by size-exclusion chromatography (SEC), peptides are derivatized with phenyl isothiocyanate. The resultant phenyl thiocarbamyl (PTC)-peptides can be resolved with high resolution and sensitivity by RP-HPLC. By comparing chromatograms of PTC derivatives from blood before and after ingestion of a peptide sample, food-derived peptide can be detected. The isolated PTC-peptide can be applied to a peptide sequencer based on the Edman degradation reaction.     

Recent developments in CE and CEC of peptides (2009-2011)

The review brings a comprehensive survey of the recent developments of high-performance electroseparation methods in capillary and microchip formats: zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography. Applications of these techniques to analysis, isolation, purification and physicochemical and biochemical characterization of peptides are described. Advances in the investigation of electromigration properties of peptides, and in the methodology of their analysis, such as sample preparation, adsorption suppression, EOF control and detection, are presented. New developments, in particular, CE and CEC modes are reported and several types of their applications to peptide analysis are described: conventional qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide relevant physicochemical characteristics of peptides are demonstrated.     

High-performance affinity chromatography

High-performance affinity chromatography is a new technique for the fast and efficient purification of biologically active molecules. It combines the biospecificity of affinity chromatography with the high speed and resolution obtained in high-performance liquid chromatography. In particular, the immobilization of ligands to different silica derivatives and their suitability for high-performance affinity chromatography are discussed.     

Ultra performance liquid chromatography (UPLC) further improves hydrogen/deuterium exchange mass spectrometry

Ultra performance liquid chromatography (UPLC) employs particles smaller than 2 microm in diameter to achieve superior resolution, speed, and sensitivity compared with high-performance liquid chromatography (HPLC). We have tested the suitability of UPLC for the analysis of deuterated peptides in hydrogen exchange mass spectrometry experiments. Superior resolution and sample throughput were obtained with UPLC versus HPLC. For highly deuterated model peptides, deuterium loss using UPLC was greater than the deuterium loss observed using a conventional HPLC system, primarily as a result of the injection requirements of the UPLC system. Partially deuterated cytochrome c peptides also lost more deuterium in UPLC versus HPLC, although the effect was not as pronounced as it was for the highly deuterated model peptides. The exceptional chromatographic aspects of UPLC make it a very attractive alternative to HPLC for hydrogen exchange mass spectrometry experiments.