Sequence-specific retention calculator. A family of peptide retention time prediction algorithms in reversed-phase HPLC: applicability to various chromatographic conditions and columns

Separation selectivity of C18 reversed-phase columns from different manufacturers has been compared to evaluate the applicability of our sequence-specific retention calculator (SSRCalc) peptide retention prediction algorithms. Three different versions of SSRCalc are currently in use: 300-A pore size sorbents (TFA as ion-pairing modifier, pH 2), 100 A (TFA, pH 2), and 100 A (pH 10), which have been applied for the separation of randomly chosen mixture of tryptic peptides. The major factor affecting separation selectivity of C18 sorbents was found to be apparent pore size, while differences in end-capping chemistry do not introduce a significant impact. The introduction of embedded polar groups to the C18 functionality increases the retention of peptides containing hydrophobic amino acid residues with polar groups: Tyr and Trp. We also demonstrate that changing the ion-pairing modifier to formic/acetic acid significantly reduces the algorithm's predictive ability, so models developed for different eluent conditions cannot be compared directly to each other.     

Characterization of open tubular capillary electrochromatography columns for the analysis of synthetic peptides using isocratic conditions.

In this paper, we report on investigations related to the performance characteristics of two different types of etched chemically (n-octadecyl- and cholesterol-) modified capillaries in the open tubular format of capillary electrochromatography (CEC) for the analysis of synthetic peptides. The results confirm that the nature of the surface chemistry used to modify the capillary wall and type of chemically bonded group employed can affect the selectivity as well as the resolution of peptide samples. The results are consistent with the participation of selective peptide interactions with the bonded phase, although other factors, such as the morphology of the capillary wall surfaces, appear to be also involved. Moreover, several surprising observations related to peptide-specific multi-zoning effects have been observed. Additional experimental variables that can also be utilized to affect the retention of peptides in this approach to OTCEC include the type and percentage of organic solvent modifier employed in the eluent and the pH of the buffer system. To evaluate the reproducibility of different batches of the n-octadecyl- and cholesterol-modified capillaries and the stability of the chemically modified surface, the OTCEC selectivity and peak shape behavior of two small basic molecules (serotonin and tryptamine) and two proteins (turkey and chicken lysozyme) were also investigated. Finally, the use of the "bubble" cell technology for creating the detector window has been shown to provide significantly higher detection sensitivity with peptides, as compared with the conventional capillary format.     

A study of the Lewis acid-base interactions of vinylphosphonic acid-modified polybutadiene-coated zirconia

Polybutadiene-coated zirconia (PBD-ZrO2) is very useful for reversed-phase separations under a wide variety of conditions. Its excellent chemical (pH = 1-13) and thermal (up to 150 degrees C) stability distinguish it from silica-based reversed phases. Just as with silica-based phases, zirconia's surface chemistry significantly influences the chromatography of certain classes of analytes. Zirconia's hard Lewis acid sites can be chromatographically problematic. Analytes such as carboxylic acids strongly interact with these sites on PBD-ZrO2 and do not elute. Addition of phosphate or other strong, hard Lewis bases to the eluent brings about elution, but the resulting peak is often tailed and broad. Typically, cationic solutes are more retained in the presence of phosphate or fluoride due to adsorption of the Lewis base additives and the concomitant development of a negative charge on the surface. This Coulombic interaction can be used to optimize selectivity, but the reversed-phase-cation-exchange retention can produce broad peaks with excessive retention. As an alternative to adding Lewis bases to the eluent, we studied the effect of permanently modifying PBD-ZrO2 by covalently attaching vinylphosphonic acid (VPA) to PBD which was predeposited in the pores of zirconia. We have investigated the chromatography of acids, bases, and small peptides on VPA-modified PBD-ZrO2 (VPA-PBD-ZrO2) and compared it to PBD-ZrO2. VPA-PBD-ZrO2 is a reversed-cation-exchange phase with properties quite different from PBD-ZrO2. The chemical stability of both phases led us to explore how low-pH (1.5-3), ultralow-pH (0), and high-pH (12) eluents effect the retention properties of these mixed-mode phases. Ultralow-pH eluents effectively separate small peptides on both phases. This approach gives lower retention, without sacrificing resolution, and much higher efficiency for small peptides than previously reported.     

Use of a mixed-mode packing and voltage tuning for peptide mixture separation in pressurized capillary electrochromatography with an ion trap storage/reflectron time-of-flight mass spectrometer detector.

A mixed-mode (reversed-phase/anion-exchange) stationary phase has been used as the capillary column packing for investigation of the separation of peptide mixtures in pressurized capillary electrochromatography (pCEC). This stationary phase contains both octadecylsilanes and dialkylamines. The amine groups of the stationary phase determine the charge density on the surface of the packing and can produce a strong and constant electroosmotic flow (EOF) at low pH. A comparison was made in terms of the capability of separating tryptic digests between the mixed-mode phase and C18 reversed phase. In addition, the constant EOF enabled the tuning of the retention and the selectivity of the separation by adjusting the mobile phase pH from 2 to 5. Furthermore, the magnitude and the polarity of the electric voltage were demonstrated to greatly influence the elution profiles of the peptides in pCEC. An ion trap storage/reflectron time-of-flight mass spectrometer was used as an on-line detector in these experiments due to its ability to provide rapid and accurate mass detection of the sample components eluting from the separation column.     

Deamidation of -Asn-Gly- sequences during sample preparation for proteomics: Consequences for MALDI and HPLC-MALDI analysis

We find that peptides containing -Asn-Gly- sequences typically show approximately 70-80% degree of deamidation after standard overnight (approximately 12 h) tryptic digestion at 37 degrees C. This emphasizes the need for more detailed information about the deamidation reaction in -Asn-Gly- sequences, in which two deamidated species are produced, one containing an aspartic acid (-Asp-Gly-) residue and the other containing an isoaspartic acid (-betaAsp-Gly-) residue. For the peptide SLNGEWR (54-60 beta-galactosidase, E. coli), all three components of the reaction mixture were separated by HPLC on C18 300-A sorbent, with trifluoroacetic acid as an ion-pairing modifier. Their intensity ratios suggested the elution order -betaAsp-/-Asn-/-Asp-, which was subsequently confirmed by MALDI MS and MS/MS analysis. The kinetics of the deamidation was studied in detail for the synthetic SLNGEWR parent using RP HPLC with UV detection. The half-life of this peptide was found to be approximately 8 h under digestion conditions. Analysis of a large pool of peptide retention data shows that the -betaAsp-/-Asn-/ -Asp- retention order is normally observed under the above conditions, especially if the original -NG- sequence is surrounded by hydrophobic amino acids. However, changing chromatographic conditions to 100-A pore size sorbents, or using formic acid as a modifier, increases the retention time of -betaAsp- relative to the -Asn-/-Asp- pair, so the order can sometimes be different.     

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

Fundamentals of capillary electrochromatography: migration behavior of ionized sample components

The mechanism of separating charged species by capillary electrochromatography (CEC) was modeled with the conditions of ideal/linear chromatography by using a simple random walk. The most novel aspect of the work rests with the assumption that in sufficiently high electric field ionized sample components can also migrate in the adsorbed state on the ionized surface of the stationary phase. This feature of CEC leads to the introduction of three dimensionless parameters: alpha, reduced mobility of a sample component with the electrosmotic mobility as the reference; beta, the CEC retention factor; and gamma, the ratio of the electrophoretic migration velocity and the velocity of surface electrodiffusion. Since the interplay of retentive and electrophoretic forces determines the overall migration velocity, the separation mechanism in CEC is governed by the relative importance of the above parameters. The model predicts conditions under which the features of the CEC system engender migration behavior that manifests itself in a relatively narrow elution window and in a gradient like elution pattern in the separation of peptides and proteins by using pro forma isocratic CEC. It is believed that such elution patterns, which resemble those obtained by the use of external gradient of the eluent, are brought about by the formation of an internal gradient in the CEC system that gave rise to concomitant peak compression. The peculiarities of CEC are discussed in the three operational modalities of the technique: co-current, countercurrent, and co-counter CEC. The results suggest that CEC, which is often called "liquid chromatography on electrophoretic platform" is an analytical tool with great potential in the separation of peptides and proteins.     

Sequence-specific retention calculator. Algorithm for peptide retention prediction in ion-pair RP-HPLC: application to 300- and 100-A pore size C18 sorbents

Continued development of a new sequence-specific algorithm for peptide retention prediction in RP HPLC is reported. Our discovery of the large effect on the apparent hydrophobicity of N-terminal amino acids produced by the ion-pairing retention mechanism has led to the development of sequence-specific retention calculator (SSRCalc) algorithms. These were optimized for a set of approximately 2000 tryptic peptides confidently identified by off-line microHPLC-MALDI MS (MS/MS) (300-A pore size C18 sorbent, linear water/acetonitrile gradient, and trifluoroacetic acid as ion-pairing modifier). The latest version of the algorithm takes into account amino acid composition, position of the amino acid residues (N- and C-terminal), peptide length, overall hydrophobicity, pI, nearest-neighbor effect of charged side chains (K, R, H), and propensity to form helical structures. A correlation with R2 approximately 0.98 was obtained for the 2000-peptide optimization set. A flexible structure for the SSRC programming code allows easy adaptation to different chromatographic conditions. This was demonstrated by adapting the algorithm (approximately 0.98 R2 value) for a set of approximately 2500 peptides separated on a 100-A pore size C18 column. The SSRCalc algorithm has also been extensively tested for a number of real samples, providing solid support for protein identification and characterization; correlations in the range of 0.95-0.97 R2 value have normally been observed.     

Dynamics of sorption of cesium radionuclides on selective ferrocyanide sorbents. Distribution of the 137Cs activity in the stationary phase

A method was suggested for studying the dynamic sorption properties of ferrocyanide sorbents toward Cs radionuclides by analyzing the radiocesium distribution in the solid phase of the sorbent. The dynamic sorption characteristics are determined by measuring the activity of successive sorbent layers after passing definite volumes of the solution. The method is efficient in analysis of the stability of various sorbents in liquid media. The sorption characteristics of the following ferrocyanide sorbents were examined: Termoksid-35 (nickel-potassium ferrocyanide on zirconium hydroxide), FNS-2 (nickel-potassium ferrocyanide on silica), and FS-2 (copper-potassium ferrocyanide on silica). In alkaline solutions, Termoksid-35 is the most stable. The effect of oxalic acid on the activity distribution in a ferrocyanide sorbent bed was studied. In alkaline solutions containing oxalic acid, copper ferrocyanide (FS-2) is the least stable. At the same time, at low pH values and in the absence of oxalic acid, it is preferable to use sorbents based on copper ferrocyanides.     

Capillary electrochromatography of proteins on an anion-exchanger column

Capillary electrochromatography (CEC) of proteins was carried out using 50-microm-i.d. fused-silica capillaries packed with 5-microm silica beads having strong anion-exchanger functions attached to hydrophilic spacers at the chromatographic surface. The siliceous microspheres and the capillary innerwall were treated first with a heterobifunctional silanizing agent and reacted subsequently with a vinyl monomer containing quaternary ammonium groups to form a "tentacular" anion exchanger. A mixture of bovine carbonic anhydrase, alpha-lactalbumin, soybean trypsin inhibitor, and ovalbumin was separated using CEC by isocratic elution in the codirectional mode with aqueous phosphate buffer, pH 7.0, containing sodium chloride. The retention mechanism of isocratic CEC for proteins on the anion-exchanger column was illustrated by the results of a study on the effect of salt concentration on the separation. The potential of CEC for protein separation with high resolution was also demonstrated by electrochromatograms of conalbumin and hemoglobin variants. The results shed light on the mechanism of protein separation by isocratic CEC, which is believed to be a combination of chromatographic retention by electrostatic interactions and electrophoretic migration. Assuming that the contributions of the two mechanisms to the overall migration velocity are additive, an electrochromatographic resolution equation was derived and compared to the resolution equation in HPLC to reveal the constituents responsible for the enhancement of resolution by CEC with respect to that in HPLC. The advantage of CEC was also examined by comparing peak capacities in CEC on an, isocratic platform with peak capacities obtained with isocratic and gradient elution HPLC.     

Temperature-Responsive Chromatography Using Poly(N-isopropylacrylamide)-Modified Silica

A new concept in chromatography is proposed that utilizes a temperature-responsive surface with a constant aqueous mobile phase. The surface of the silica stationary phase in high-performance liquid chromatography (HPLC) has been modified with temperature-responsive polymers to exhibit temperature-controlled hydrophilic/hydrophobic changes. Poly(N-isopropylacrylamide) (PIPAAm) was grafted onto (aminopropyl)silica using an activated ester-amine coupling method. These grafted silica surfaces show hydrophilic properties at lower temperatures which, as temperature increases, transform to hydrophobic surface properties. The elution profile of five mixed steroids on an HPLC column packed with this material depends largely on the temperature of the aqueous mobile phase. Retention times increase with increasing temperature without any change in the eluent. Changes in the retention times of hydrophobic steroids were larger than those for hydrophilic steroids. The temperature-responsive interaction between PIPAAm-modified silica and these steroids is proposed to result from changes in the surface properties of the HPLC stationary phase by the transition of hydrophilic/hydrophobic surface-grafted IPAAm polymers. We demonstrate a novel and useful new chromatography system in which surface properties and the resulting function of the HPLC stationary phase are controlled by external temperature changes. This method should be effective in biological and biomedical separations of peptides and proteins using only aqueous mobile phases.     

镉离子印迹硅胶的制备与吸附性能

采用表面印迹技术和溶胶-凝胶法,以Cd(Ⅱ)离子作为印迹离子,硫氰基丙基三甲氧基硅烷为功能分子,环氧氯丙烷为交联剂,在硅胶表面制备Cd(Ⅱ)离子印迹聚合物(IIP-TCPTS/SiO2),并利用平衡吸附法研究了聚合物吸附性能和选择识别能力。结果表明,最大吸附量为16.7 mg/g;20 min即可达到吸附平衡;当pH值在5.4~7.8范围内,印迹聚合物保持了较好的吸附容量;印迹聚合物对Cd(Ⅱ)离子具有较强的选择性识别能力;重复使用时性能稳定。     

Geochemical aspects of environmentally safe conservation of liquid radioactive waste

Physicochemical features of the sorption of Sr, Cs, and U radionuclides on natural mineral sorbents (montmorillonites of Na and Ca type, kaolinites, illites) were studied. The main processes responsible for binding and retention of radionuclides are ion exchange and formation of complexes on the mineral surface. The influence of pH, salt composition of the solution, specific surface area of the sorbent, and its pore size on the radionuclide immobilization efficiency was examined.     

Behavior of cation-exchange materials in capillary electrochromatography

The behavior of a strong, cation-exchange material (propanesulfonic acid, SCX) has been studied in capillary electrophoresis (CE) and capillary electrochromatography (CEC) by the use of coated and packed capillaries. In aqueous electrolytes, the SCX-coated capillary showed a far more consistent electroosmotic flow over the pH range 3.6-10.5, compared to untreated fused silica. However, in similar electrolytes containing 80% (v/v) acetonitrile, both coated and untreated capillaries performed similarly, casting doubts upon the stability of the SCX coating. The effect of voltage and mobile-phase parameters such as pH, ionic strength, and organic content was studied in CEC for both 3-μm SCX and C(18) packing materials, and the results were compared in terms of linear velocities, currents, and conductivities. Only at pH 5 and below was a higher EOF velocity than expected observed for the SCX column. In accordance with theory, the EOF was seen to increase with decreasing ionic strength for the C(18) column. However, for the SCX column, this was not the case: the EOF showed a general reduction as the ionic strength was decreased. The greatest anomaly was observed on changing the acetonitrile composition: the EOF showed a consistent decline with increasing organic, whereas the EOF in both the open capillary and C(18) column decreased and then started to rise with acetonitrile contents above 70% (v/v).     

Retention behavior of ionizable isomers in reversed-phase liquid chromatography: a comparative study of porous graphitic carbon and octadecyl bonded silica

The retention behaviors of 36 positional isomers of ionizable substituted benzene compounds have been compared on two different packing materials: porous graphitic carbon (PGC) and octadecyl bonded silica (ODS) using 35% aqueous acetonitrile as the mobile phase. The effect of the mobile phase pH on the solute retention was studied over a range of pH values from pH 2.0 to 7.0. The retention as a function of pH was modeled using equations based on solute ionization. With PGC, the theoretical equations fitted the observed retention data for each class of solute, indicating that the retention mechanism was uniform over the whole pH range. However, with ODS, only the acidic solutes showed agreement with the theoretical model; for the amine-containing compounds, serious deviations from the theory were observed, suggesting that strongly acidic silanols gave added retention at low mobile phase pH. Overall, PGC demonstrated a higher selectivity toward positional isomers than ODS. This was attributed to the greater steric discriminating ability arising from the flat surface of the PGC compared with the more fluid nature of the ODS bonded phase.     

Open tubular capillary electrochromatography of synthetic peptides on etched chemically modified columns

Two sets of peptides, each having structurally similar amino acid sequences, have been investigated by capillary electrochromatography (CEC) using etched chemically modified capillaries as the separation medium. In comparison to gradient RP-HPLC, the resolving power of the described CEC methods has been found to be superior. A number of variables have been examined with respect to optimization of the separation of these closely related peptides with several different etched chemically modified capillaries. These experimental variables included the nature of the bonded moiety, the pH, the organic modifier type, and the amount of organic modifier in the buffer electrolyte. Systematic variation of these parameters results in significant changes in the migrational behavior of the investigated peptides and provides important insight into the underlying molecular separation processes that prevail in open tubular CEC. Moreover, under optimized conditions, efficient separations characterized by highly symmetrical peaks were achieved. In addition, this study has permitted the long-term stability as well as the short-term and long-term reproducibility of the etched chemically modified capillaries to be documented.     

Influence of pH on retention in linear organic modifier gradient RP HPLC

The purpose of this work was to examine the influence of pH on retention of analytes during the linear organic modifier gradient in RP HPLC. We started from the general theory of combined pH/organic modifier gradient and provided an explicit however approximate solution relating gradient retention time and the pH of the eluent. The accuracy of the proposed model was tested by its ability to describe an experimental data set that comprised retention times for a series of monoprotic acids and bases obtained at different pH and for different gradient durations. The basic analytical properties of the equation relating retention time and pH were determined, such as the inflection point and range of retention times. The use of the pH at inflection point as a measure of p K a was discussed and compared to the chromatographic p K a obtained by a fitting to the proposed model. In conclusion, the work provides theoretical results that augment the knowledge on the impact of pH on the gradient retention.     

Fully packed capillary electrochromatographic microchip with self-assembly colloidal silica beads

A fully packed capillary electrochromatographic (CEC) microchip showing improved solution and chip handling was developed. Microchannels for the CEC microchip were patterned on a cyclic olefin copolymer substrate by injection molding and packed fully with 0.8-microm monodisperse colloidal silica beads utilizing a self-assembly packing technique. The silica packed chip substrate was covered and thermally press-bonded. After fabrication, the chip was filled with buffer solution by self-priming capillary action. The self-assembly packing at each channel served as a built-in nanofilter allowing quick loading of samples and running buffer solution without filtration. Because of a large surface area-to-volume ratio of the silica packing, reproducible control of electroosmotic flow was possible without leveling of the solutions in the reservoirs resulting 1.3% rsd in migration rate. The capillary electrophoretic separation characteristics of the chip were studied using fluorescein isothiocyanate (FITC)-derivatized amino acids as probe molecules. A mixture of FITC and four FITC-derivatized amino acids was successfully separated with 2-mm separation channel length.     

Supercritical fluid generated stationary phases for liquid chromatography and capillary electrochromatography

Chromatographic silica-bonded stationary phases have been prepared using supercritical CO(2) as the reaction medium. (29)Si solid-state NMR spectra of the generated bonded silica phases show unreacted silica species Q(3) and Q(4), alongside important resonances for surface-bound ligands, T(1), T(2), and T(3). Initially, a fluorinated octyl silica (C(8)) phase was produced, by reacting (1)H,(1)H,(2)H,(2)H-perfluorooctyltriethoxysilane with silica particles (3 microm) in sc-CO(2) at 60 degrees C and 450 atm for 3 h. In-house-packed LC columns of this fluorinated sc-C(8) silica phase yielded typical reversed-phase behavior when a standard test mix of benzamide (k' = 1.03), benzophenone (k' = 8.11), and biphenyl (k' = 14.92) was eluted. When packed into fused-silica capillaries for CEC, this fluorinated sc-C(8) silica phase gave linear plots of log k' versus percentage MeOH for benzophenone and biphenyl and, in contrast to octyl or octadecyl silica phases, displayed selectivity for aromatic thioureas when chromatographed among a series of synthetic organic thiourea test solutes. Similarily, an octadecyl silica phase (sc-C(18) silica) was prepared by reaction of n-octadecyltriethoxysilane in sc-CO(2), packed at 9500 psi and examined by LC. The sc-C(18) silica LC column yielded high column efficiency (up to 141 000 N/m (fluorene)) and excellent asymmetry factors (1.06, fluorene) without resource to end-capping. Following a second silylating or end-capping step using hexamethyldisilazane in sc-CO(2), sc-end-capped sc-C(18) silica phases elute N,N-DMA before toluene and the toluidine isomers as a single peak, indicating lowered silanol activity according to the Engelhardt test. A rapid separation of the important pharmaceutical substances, ketoprofen, naproxen, fentoprofen, and ibuprofen, on an sc-end-capped sc-C(18) silica phase is also shown.     

Cross-linked thermoresponsive anionic polymer-grafted surfaces to separate bioactive basic peptides

Cross-linked, thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) [poly(IPAAm-co-AAc-co-tBAAm)] thin hydrogel layers on silica beads were used as new column matrix modifiers for LC separation of basic bioactive peptides, angiotensin subtypes I, II, and III. Terpolymer poly(IPAAm-co-AAc-co-tBAAm) showed both phase transition and apparent carboxylate pKa shifts in water, depending on temperature. Polymer-grafted silica bead surfaces exhibited simultaneous thermally modulated changes in hydrophilic/hydrophobic properties and charge densities. More effective separation of angiotensin peptide subtypes was achieved on columns of these terpolymer thin hydrogel grafted surfaces, as compared to an uncharged control binary copolymer of IPAAm and tBAAm. Although hydrophobic interactions effect separation of angiotensin subtypes, combined electrostatic and hydrophobic interaction resulted in more pronounced retention. At temperature below the terpolymer phase transition, hydrophobic interactions predominated, and minimal changes in electrostatic interactions were supported by little shift in the apparent AAc carboxylate pKa values. Above the phase transition temperature, electrostatic interactions were dramatically reduced as a result of the decreased charge densities of the polymer grafted surfaces. Therefore, peptide retention times were also reduced, exhibiting a maximum at near 30-35 degrees C. Interestingly, column retention behavior of angiotensins is dramatically modulated by applied step temperature gradients. Thermoresponsive surface property alteration is a very rapid, reversible phenomenon, allowing step temperature gradients on thermoresponsive columns to enable the analogous performance advantages as gradient elution in reversed-phase HPLC. More importantly, injected peptides were recovered completely from the columns from calculation of peak area. In conclusion, these anionic thermoresponsive polymer-modified surfaces are good candidates for improved separation of bioactive peptides under exclusively aqueous conditions.