Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.     

Effect of anionic ion-pairing reagent concentration (1-60 mM) on reversed-phase liquid chromatography elution behaviour of peptides

The homologous series of volatile perfluorinated acids-trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--continue to be excellent anionic ion-pairing reagents for reversed-phase high-performance liquid chromatography (RP-HPLC) after more than two decades since their introduction to this field. It was felt that a thorough, step-by-step re-examination of the effects of anionic ion-pairing reagents over a wide concentration range on RP-HPLC peptide elution behaviour is now due, particularly considering the continuing dominance of such reagents for peptide applications. Thus, RP-HPLC was applied over a range of 1-60 mM phosphoric acid, TFA, PFPA and HFBA to two mixtures of 18-residue synthetic peptides containing either the same net positive charge (+4) or varying positive charge (+1, +2, +3, +4). Peptides with the same charge are resolved very similarly independent of the ion-pairing reagent used, although the overall retention times of the peptides increase with increasing hydrophobicity of the anion: phosphate < TFA- < PFPA- < HFBA-. Peptides of differing charge move at differing rates relative to each other depending on concentration of ion-pairing reagents. All four ion-pairing reagents increased peptide retention time with increasing concentration, albeit to different extents, again based on hydrophobicity of the anion, i.e., the more hydrophobic the anion, the greater the increase in peptide retention time at the same reagent concentration. Interestingly, phosphoric acid produced the best separation of the four-peptide mixture (+1 to +4 net charge). In addition, concentrations above 10 mM HFBA produced a reversal of the elution order of the four peptides (+1 < + 2 < + 3 < + 4) compared to the elution order produced by the other three reagents over the entire concentration range (+4 < + 3 < + 2 < + 1).     

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.     

Analysis of sulfonated compounds by reversed-phase ion-pair chromatography—Mass spectrometry with on-line removal of non-volatile tetrabutyl ammonium ion-pairing agents

Summary An ion-pair reversed-phase high performance liquid chromatography method with tetrabutyl ammonium salts as ion-pairing agents and mass spectrometric detection has been developed for sulfonated compounds with special focus on structural identification. A cation exchange suppressor cartridge placed between the UV detector and the ion source of the mass spectrometer completely removed the non-volatile ion-pairing agent resulting in excellent conditions for both electrospray ioniziation and atmospheric pressure chemical ioniziation. Peak broadening caused by the dead volume of the suppressor is negligible. The application range of this method is demonstrated by the structural identification of individual compounds within a complex mixture of model substances consisting of sulfonated aromatics, textile dyes and detergents with as many as four sulfonic acid groups, all largely differing from each other in their structural properties. The influence of the ion-pairing agent's counter ion on retention behaviour is also discussed.     

High-performance size-exclusion chromatography of polypeptides on a TSK G2000SW column in acidic mobile phases

Polypeptides subjected to high-performance liquid chromatography on a TSK G2000SW column in acidic mobile phases of low ionic strength underwent a combination of size exclusion and ion exclusion from the matrix. The distribution coefficient (Kd) of each polypeptide was dependent upon the concentration of ion-pairing agent present and increased as the concentration of ion-pairing agent increased. The fractionation range of the column could thus be manipulated by altering the concentration of acid in the mobile phase. The nature of the ion-pairing acid, in terms of hydrophilic or hydrophobic ion pairing, also had an effect on the chromatography, such that hydrophobic ion-pairing agents lead to increased Kd values, especially in the case of the most basic polypeptides. To account for these results it is proposed that the TSK GSW matrix is cationic at low pH, and that ion pairing suppresses the ion exclusion experienced by cationic polypeptides under these conditions.     

Separation of metalloporphyrins from metallation reactions by liquid chromatography and electrophoresis

The analytical separation of the indium and manganese complexes of three synthetic, meso-substituted, water-soluble porphyrins from their respective free bases in metallation reaction mixtures is described. The ligands tetra-3N-methylpyridyl porphyrin, tetra-4N-methylpyridyl porphyrin and tetra-N,N,N-trimethylanilinium porphyrin are complexed with In (III) and Mn (III) and are separated from residual free base by high-performance liquid chromatography (HPLC) in acidic conditions with gradient elution on ODS bonded stationary phase. Electrophoretic separation is achieved on both cellulose polyacetate strips and polyacrylamide tube gels under basic conditions. Although analytical separations can be achieved by both HPLC and electrophoresis, only HPLC is suitable for the development of preparative scale separations. Column chromatography, ion-pairing and ion-suppression HPLC techniques fail to separate such highly charged and closely related aromatic compounds.     

The effects of pH, ionic strength and buffer concentration of mobile phase onR F of acidic compounds in ion-pair TLC

Summary The effects of some factors important in ion-pair, high-performance liquid chromatography were studied in ion-pair, thin-layer chromatography. Tetramethyl and cetyltrimethylammonium salts were used as ion-pairing reagents. As stationary phases, silica gel and chemically bonded reversed-layers (C₁₈) were used. Layers were impregnated with ion-pairing reagent prior to chromatography. In some cases the stationary phase was treated with buffer at different concentrations. The mobile phase contained methanol and water, in one set of experiments buffer, salt for adjustment of ionic strength and ion-pairing reagent were added. The migration behaviour of different benzoic acids was studied. Several problems of ion-pair thin-layer chromatography are discussed. Passed away on 13^th of April, 1998 Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Capillary electrophoresis of cationic random coil peptide standards: effect of anionic ion-pairing reagents and comparison with reversed-phase chromatography

The present study compares a charge/hydrophobicity capillary electrophoresis (CE) approach to reversed-phase high-performance liquid chromatography (RP-HPLC) for the separation of three series of four synthetic, random coil peptide standards. Each series has peptides of the same positive charge (+1, +2 and +3 series) and length but differing in hydrophobicity. Complete resolution of the 12 peptides was achieved via a novel CE approach: a capillary zone electrophoresis (CZE) mode effected a separation of identically charged peptides; within each charged group of peptides, the addition of perfluorinated acid anionic ion-pairing reagents allowed resolution of the peptides through a mechanism based on peptide hydrophobicity which we have termed ioninteraction (II)-CZE. The peak capacity and peptide resolution of this CE approach was superior to that of RP-HPLC and stresses an important role for CE for peptide/proteomic applications.     

Effect of anionic ion-pairing reagent hydrophobicity on selectivity of peptide separations by reversed-phase liquid chromatography

Despite the continuing dominance of trifluoroacetic acid (TFA) as the anionic ion-pairing reagent of choice for peptide separations by reversed-phase high-performance liquid chromatography (RP-HPLC), we believe that a step-by-step approach to re-examining the relative efficacy of TFA compared to other ion-pairing reagents is worthwhile, particularly for the design of separation protocols for complex peptide mixtures, e.g., in proteomics applications. Thus, we applied RP-HPLC in the presence of different concentrations of anionic ion-pairing reagents - phosphoric acid, TFA, pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA)--to a mixture of three groups of four 10-residue peptides, these groups containing peptides of +1, +3 or +5 net charge. Overall separation of the 12-peptide mixture improved with increasing reagent hydrophobicity (phosphate- < TFA- < PFPA- < HFBA-) and/or concentration of the anion, with reagent hydrophobicity having a considerably more pronounced effect than reagent concentration. HFBA, in particular, achieved an excellent separation at a concentration of just 10 mM, whereby the peptides were separated by charged groups (+1 < +3 < +5) and hydrophobicity within these groups. There was an essentially equal effect of reagent hydrophobicity and concentration on each positive charge of the peptides, a useful observation for prediction of the effect of varying counterion concentration hydrophobicity and/or concentration during optimization of peptide purification protocols. Peak widths were greater for the more highly charged peptides, although these could be decreased significantly by raising the acid concentration; concomitantly, peptide resolution increased with increasing concentration of ion-pairing reagent.     

Context-dependent effects on the hydrophilicity/hydrophobicity of side-chains during reversed-phase high-performance liquid chromatography: Implications for prediction of peptide retention behaviour

The present study set out to investigate whether observed relative hydrophilicity/hydrophobicity values of positively charged side-chains (with Lys and Arg as representative side-chains) or hydrophobic side-chains (with Ile as the representative side-chain) were context-dependent, i.e., did such measured values vary depending on characteristics of the peptides within which such side-chains are substituted (overall peptide hydrophobicity, number of positive charges) and/or properties of the mobile phase (anionic counterions of varying hydrophobicity and concentration)? Reversed-phase high-performance liquid chromatography (RP-HPLC) was applied to two series of four synthetic peptide analogues (+1, +2, +3 and +4 net charge), the only difference between the two peptide series being the substitution of one hydrophobic Ile residue for a Gly residue, in the presence of anionic ion-pairing reagents of varying hydrophobicity (HCOOH approximately H3PO4 < TFA < PFPA < HFBA) and concentration (2-50 mM). RP-HPLC of these peptide series revealed that the relative hydrophilicity of Lys and Arg side-chains in the peptides increased with peptide hydrophobicity. In addition the relative hydrophobicity of Ile decreased dramatically with an increase in the number of positive charges in the peptide, this hydrophobicity decrease being of greater magnitude as the hydrophobicity of the anionic ion-pairing reagent increased. These results have significant implications in the prediction of peptide retention times for proteomic applications.     

Peptide mapping with mobile phases of intermediate pH value using capillary reversed-phase high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

This investigation describes the separation of tryptic peptides by capillary reversed-phase high-performance liquid chromatography (RP-HPLC) with eluents in the intermediate pH range, followed by in-line electrospray ionisation tandem mass spectrometry (ESI-MS/MS) analysis. For these purposes, gradient elution procedures with an aqueous eluent containing 20 mM ammonium formate, and an increasing content of acetonitrile or methanol, were employed. Compared to the analysis of the same tryptic peptides under low-pH conditions with an ion-pairing reagent, the increase in the pH with the 20 mM ammonium formate mobile phase led to significant changes in both peptide retention to the reversed-phase column and the collision-induced dissociation at the MS/MS stage as a consequence of the changes in the physico-chemical properties of these peptides, such as their overall charge, polarity and relative hydrophobicity. Thus, improved selectivity for the peptide separation and favourable tandem mass spectrometry analysis could be obtained with eluents in this intermediate pH range. The number of tryptic peptides identified by the new approach for the proteins investigated were significantly higher than that obtained by the conventional low-pH methods. Moreover, analysis of protein digests at very low concentrations was also performed under both acidic and intermediate pH conditions and similar improvements in selectivity and MS/MS detection limits were observed, i.e. identification of more distinct peptides and higher sequence coverage of the protein was obtained when eluents of intermediate pH were employed. This study therefore highlights the potential of conducting peptide mapping in the intermediate pH range to achieve more reliable and sensitive protein identifications with capillary RP-HPLC–ESI-MS/MS.     

Reversed-phase high-performance liquid chromatography of amphoteric beta-lactam antibiotics: effects of columns, ion-pairing reagents and mobile phase pH on their retention times

The separation of five amino beta-lactam antibiotics by reversed-phase high-performance liquid chromatography was studied as an insight into their retention behaviour. These five amphoteric compounds are cephradine, cephalexine, cefaclor, ampicillin and amoxicillin. Both octadecylsilane-bonded silica (C18) columns and phenyl-bonded silica (phenyl) columns were used, with mobile phase pH values between 2.5 and 7.4. In the absence of ion-pairing reagents the retention times for all the five compounds were the shortest at pH 4-6. The phenyl column was found to improve the separation between cephradine and ampicillin at pH values lower than 3, when these two compounds appeared as fused peaks on the C18 on C18 columns, with mobile phases both with and without ion-pairing reagents, were compared. The addition of 0.005 or 0.02 M tetraethylammonium acetate to the mobile phase did not result in significant ion-pair formation, except at pH values higher than 5.5. A strong ion-pairing effect was obtained at pH values higher than 6 with 0.005 or 0.02 M tetrabutylammonium phosphate, and the retention was decreased at pH values lower than 4. On the other hand, 0.005 M heptanesulphonic acid exhibited an ion-pair retention effect at pH values lower than 5. The molecular structures and pK(a) values were used to account for the retention behaviour of these antibiotics in the various mobile phases.     

Micellar electrokinetic chromatography separations of dynorphin peptide analogs

Prodynorphin is a precursor that has multiple cleavage sites to release various dynorphin opioid peptides. The dynorphin analogs used in this study have 18 amino acid residues. A series of dynorphin-like peptides, differing by a single residue (alanine substitution) were assembled by Fmoc solid-phase procedures and purified by preparative high performance liquid chromatography (HPLC). Separation of the Ala-scan dynorphin analogs was investigated by micellar electrokinetic chromatography (MEKC) employing anionic, cationic and zwitterionic surfactants. The role of electrostatic and hydrophobic forces in analyte-surfactant interactions is discussed with respect to the observed elution patterns. Separation of all dynorphin analogs by MEKC using a zwitterionic surfactant shows this technique to be powerful for separating closely related peptide species. It also demonstrates the potential for using MEKC for the prescreening of peptide libraries to determine their biological activity toward specific receptors. Results from the separation of dynorphin analogs by free solution and ion-pairing capillary electrophoresis are also presented.     

Stochastic theory of size exclusion chromatography by the characteristic function approach

A general stochastic theory of size exclusion chromatography (SEC) able to account for size dependence on both pore ingress and egress processes, moving zone dispersion and pore size distribution, was developed. The relationship between stochastic-chromatographic and batch equilibrium conditions are discussed and the fundamental role of the 'ergodic' hypothesis in establishing a link between them is emphasized. SEC models are solved by means of the characteristic function method and chromatographic parameters like plate height, peak skewness and excess are derived. The peak shapes are obtained by numerical inversion of the characteristic function under the most general conditions of the exploited models. Separate size effects on pore ingress and pore egress processes are investigated and their effects on both retention selectivity and efficiency are clearly shown. The peak splitting phenomenon and peak tailing due to incomplete sample sorption near to the exclusion limit is discussed. An SEC model for columns with two types of pores is discussed and several effects on retention selectivity and efficiency coming from pore size differences and their relative abundance are singled out. The relevance of moving zone dispersion on separation is investigated. The present approach proves to be general and able to account for more complex SEC conditions such as continuous pore size distributions and mixed retention mechanism.     

Retention behaviour of an homologous series of oligodeoxythymidilic acids using reversed-phase ion-pair chromatography

Summary The retention behaviour of an homologous series of phosphorylated oligodeoxythymidylic acid (pd(T)_5–18) oligonucleotides was studied using reversed-phase ion-pair chromatography with isocratic elution conditions. The effects of temperature, pH, eluent ionic strength, percentage organic modifier, concentration and alkyl chain length of the ion-pairing reagent were investigated. The retention behaviour was generally explicable by current theoretical models of ion-pair chromatography. However, the marked effect of mobile phase pH on the retention of the oligonucleotides was unexpected, and this was ascribed to the presence of ionisable residual silanols on the surface of the reversed-phase packing material.     

Heparin-like glycosaminoglycan/amine salt-bridge interactions: a new potential tool for HLGAGs analysis using mass spectrometry

Characterization of glycosaminoglycans poses a challenge for current analytical techniques, as they are highly acidic, polydisperse and heterogeneous compounds. The purpose of this study is the separation and analysis of a partially depolymerized heparin-like glycosaminoglycan by on-line ion-pairing reversed-phase high-performance liquid chromatography/electrospray mass spectrometry. The gas-phase behavior of two synthesized glycosaminoglycans has been investigated. Dibutylamine was found to be the best suited ion-pairing reagents for mass spectrometry analysis. The optimized ion-pairing conditions provide reproducible and easily interpretable electrospray mass spectra in both negative and positive ESI modes. The glycosaminoglycans are detected as a non-covalent complex with amines. In fact, the observed ionic species and their gas-phase dissociation under CID conditions revealed the presence of salt bridge interactions in the gas phase.     

Chromatographic resolution of tryptophan enantiomers with L-Leu-L-Leu-L-Leu peptide effects of mobile phase composition and chromatographic support

Tryptophan enantiomers have been separated by zwitterion pair chromatography using L-leucine-L-leucine-L-leucine peptide as the zwitterion pairing agent. The peptide ligand is adsorbed onto an octadecylsilane support with excess ligand present in bulk solution. This article examines the roles of the hydrophobic matrix and the mobile phase components on tryptophan enantiomer binding and resolution. Capacity factors and enantioselectivites are given for both hydrophobic and hydrophilic matrices using mobile phases containing Leu-Leu-Leu peptide and/or salt. A decrease in selectivity upon the addition of mobile phase salt suggests that quadrupolar ion-pairing contributes to chiral recognition. Results indicate that binding is significantly reduced and separation is not achieved when Leu-Leu-Leu is coupled onto cross-linked or polymerized hydrophilic resins as well as onto macroporous polystyrene resin. However, resin-immobilized Leu-Leu-Asp-Leu-Leu-Leu, Leu-Leu-Glu-Leu-Leu-Leu, and Leu-Leu-Leu-Glu-Leu-Leu peptides, with ion-pairing sites designed to mimic the Leu-Leu-Leu-saturated C18 support, also do not resolve tryptophan enantiomers. This suggests the Leu-Leu-Leu structure is critical for enantiomer resolution. Because D- and L-tryptophan are separated in the absence of bulk Leu-Leu-Leu, chiral discrimination is believed to occur at the surface of the octadecylsilane support.     

Investigation of the factors that induce analyte peak splitting in capillary electrophoresis

Peak splitting has a detrimental effect on analyses by capillary electrophoresis. Many papers have reported it and several mechanisms have been proposed to explain the phenomenon. We investigated the electrophoretic behavior of an amphoteric analyte, levodopa, in phosphate buffer and observed a peak splitting phenomenon at moderate sample concentrations and under general analytical conditions, even without organic solvent. The dependence of effective mobility on pH was taken into account and pKa values of 2.30, 8.11, and 9.92 were obtained for levodopa. Then, we constructed pH-dependent distribution diagrams of levodopa and phosphate species present in aqueous solution and proposed that the most relevant factors contributing to peak splitting are the presence of ionizable groups in the analyte molecule and the occurrence of ionization, yielding charged species which interacted with buffer electrolyte species in a definite pH range to form complexes. This result is different from those presented in the literature and broadens our understanding of amphoteric analyte peak splitting.     

Studies on the degradation of blue gel pen dyes by ion-pairing high performance liquid chromatography and electrospray tandem mass spectrometry

Ion-pairing high performance liquid chromatography (IP-HPLC) was utilized to monitor the composition changes of blue gel pen ink entries on paper stored in different light conditions and natural environment. The chromatographic conditions were optimized by comparing the separation efficiencies of the blue gel pen inks using a series of ion-pairing reagents, including ammonium carbonate, ammonium acetate, triethylamine acetate, tributylamine acetate, tetrabutylammonium bromide and dihexylammonium acetate. It has been found that tributylamine acetate was a suitable ion-pairing reagent for separation of the inks on the common C18 column. The analysis results of the ink entries on paper in different aging conditions showed that the tendency of composition change in natural aging condition was similar with those in fluorescent light and UV light conditions, respectively. One main component dye of the blue gel pen ink, Acid Blue 9, and its degradation products were identified by ion-pairing high performance liquid chromatography coupled with electrospray tandem mass spectrometry. The results showed that the main degradation products originated from the Acid Blue 9. It gave a reasonable explanation for the changing rules of the relative content of the dyes in the blue gel pen ink. The results obtained can provide scientific evidences for dating of the blue gel pen ink entries on documents.     

Simple and Rapid Determination of Denaturants in Alcohol Formulations by Hydrophilic Interaction Chromatography

A hydrophilic interaction chromatography (HILIC) technique has been developed and validated for determination of common denaturants (denatonium benzoate, crystal violet and methylene blue) in denaturated alcohol formulations. Among the three different polar stationary phases (i.e., aminopropyl, cyanoethyl and silica) studied the cyanoethyl phase provided much stronger retention for the organic cations. It was shown that high efficiencies were reached only with anionic ion-pairing reagent that reduces the interactions with the silanol groups. The anion ion-pairing strength under HILIC conditions was: acetate < formate < trifluoroacetate < perchlorate. This study also investigated the effect of various experimental factors on the retention of the cyanoethyl stationary phase, such as acetonitrile content, pH and ion-pairing anion concentration in the mobile phase. The separation of three denaturants was achieved in about 8 min with a mobile phase containing 60% (v/v) acetonitrile and 10 mmol L⁻¹ HClO4. The proposed method was validated and applied to the determination of danaturating agents in various Lithuanian denaturated alcohol formulations.