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.     

Practical implementation of 2D HPLC scheme with accurate peptide retention prediction in both dimensions for high-throughput bottom-up proteomics

We describe the practical implementation of a new RP (pH 10 - pH 2) 2D HPLC-ESI/MS scheme for large-scale bottom-up analysis in proteomics. When compared to the common SCX-RP approach, it provides a higher separation efficiency in the first dimension and increases the number of identified peptides/proteins. We also employed the methodology of our sequence-specific retention calculator (SSRCalc) and developed peptide retention prediction algorithms for both LC dimensions. A diverse set of approximately 10,000 tryptic peptides from the soluble protein fraction of whole NK-type cells gave retention time versus hydrophobicity correlations, with R (2) values of 0.95 for pH 10 and 0.945 for pH 2 (formic acid) separation modes. The superior separation efficiency and the ability to use retention prediction to filter out false-positive MS/MS identifications gives promise that this approach will be a method of choice for large-scale proteomics analyses in the future. Finally, the "semi-orthogonal" separation selectivity permits the concatenation of fractions in the first dimension of separation before the final LC-ESI MS step, effectively cutting the analysis time in half, while resulting in a minimal reduction in protein identification.     

Thermodynamic interpretation of retention equilibrium in reversed-phase liquid chromatography based on enthalpy-entropy compensation

The fundamentals of the retention equilibrium in reversed-phase liquid chromatography (RPLC) are studied on the basis of enthalpy-entropy compensation (EEC). First, retention data were acquired and the influence of the nature of the compounds, organic solvent modifier, and temperature on these data was assessed. Then, the data were analyzed according to the four different methods proposed by Krug et al., and an EEC was formally established. Linear correlations were observed between the logarithm of the adsorption equilibrium constants under the different RPLC conditions, suggesting linear free energy relationships (LFERs). Finally, the variations of the retentions with the experimental conditions are shown to be quantitatively explained by a new model based on EEC. This model affords a comprehensive interpretation of the variations of retention originating from changes of either one parameter alone or several simultaneously. The slope and intercept of the LFER that relates two equilibrium systems are accounted for by the new model. The parameters of this model are the changes of enthalpy and entropy associated with the retention, the compensation temperatures, and the experimental conditions.     

Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage

The extracellular matrix of dense, avascular tissues presents a barrier to entry for polymer-based therapeutics, such as drugs encapsulated within polymeric particles. Here, we present an approach by which polymer nanoparticles, sufficiently small to enter the matrix of the targeted tissue, here articular cartilage, are further modified with a biomolecular ligand for matrix binding. This combination of ultrasmall size and biomolecular binding converts the matrix from a barrier into a reservoir, resisting rapid release of the nanoparticles and clearance from the tissue site. Phage display of a peptide library was used to discover appropriate targeting ligands by biopanning on denuded cartilage. The ligand WYRGRL was selected in 94 of 96 clones sequenced after five rounds of biopanning and was demonstrated to bind to collagen II alpha1. Peptide-functionalized nanoparticles targeted articular cartilage up to 72-fold more than nanoparticles displaying a scrambled peptide sequence following intra-articular injection in the mouse.     

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.     

Thermodynamic prediction of thermal diffusivity and thermal conductivity in Mg-Zn-La/Ce system

Basic thermal properties and mechanical properties are critical parameters for the structural magnesium alloys.Solute atoms and second phases can improve mechan...     

Separation orthogonality in temperature-programmed comprehensive two-dimensional gas chromatography

In a comprehensive two-dimensional gas chromatograph, a thermal modulator serially couples two columns containing dissimilar stationary phases. The secondary column generates a series of high-speed secondary chromatograms from the sample stream formed by the chromatogram eluting from the primary column. This series of secondary chromatograms forms a two-dimensional gas chromatogram with peaks dispersed over a retention plane rather than along a line. The method is comprehensive because the entire primary column chromatogram is transmitted through the secondary column with fidelity. One might expect that a two-dimensional separation in which both dimensions are basically the same technique, gas chromatography, would be inefficient because the two dimensions would behave similarly, generating peaks whose retentions correlate across dimensions. Applying a temperature program to the two columns, however, can tune the separation to eliminate this inefficiency. The temperature program reduces the retentive power of the secondary column as a function of progress of the primary chromatogram such that the retention mechanism of the primary column is eliminated from the second dimension. Retention of a substance in the second dimension is then determined by the difference in its interaction with the two stationary phases. Retention times in the second dimension then fall within a fixed range, and the whole retention plane is accessible. In a properly tuned comprehensive two-dimensional chromatogram, retention times in the two dimensions are independent of each other, and the two-dimensional chromatogram is orthogonal. Orthogonality is important for two reasons. First, an orthogonal separation efficiently uses the separation space and so has either greater speed or peak capacity than nonorthogonal separations. Second, retention in the two dimensions of an orthogonal chromatogram is determined by two different and independent mechanisms and so provides two independent measures of molecular properties.     

Creep failure time prediction of polymers and polymer composites

A theoretical approach for the prediction of creep rupture time of polymers and polymer composites is analyzed in the present work. This analysis takes into account the viscoelastic path at small strains and the viscoplastic path at higher stresses. The calculation of the rate of creep strain is based on a thermally activated rate process, while the emergence and growth of plastic strain, with increasing creep time, is also taken into account. When the accumulated strain attains values, high enough to lead to failure, its slope versus time exhibits an abrupt change. At this specific time, the creep rate function in respect to time appears a minimum. The creep failure time is defined as the time where the creep rate takes its minimum value. The model has been tested for various types of polymeric materials, as well as for polymer composites. Once the model parameters are estimated from short time creep strain data, then it was proved to successfully predict the creep failure time at a variety of stress levels, for all material types examined.     

Importance of monovalent ions on water retention capacity of superabsorbent polymer in cement-based solutions

The effects of various ions in cement-based solutions on the water retention capacity and ion absorbency of superabsorbent polymers (SAPs) are studied herein. It is verified that the retention capacity of the SAP is reduced due to the absorbed Ca²⁺ from solution, and the SAP releases monovalent cations such as Na⁺ into the solution upon absorption of Ca²⁺. Importantly, it was determined that non-multivalent cations in solutions play a critical role in the retention capacity of SAPs. As the total ion concentration (TIC) of the solution increases, the retention capacity improves. Higher TIC yields a weaker osmotic pressure, which reduces the driving force for the initial absorption of SAP. Therefore, the amount of the Ca²⁺ absorbed in the SAP decreases and the retention capacity improves. Based on the accurately measured ionic characteristics during absorption and desorption, the complicated retention phenomenon and its ionic dependence in cement-based solutions are clearly understood.     

Thermodynamic similarity and prediction of the properties and characteristics of substances and processes

Principles of the theory of thermodynamic similarity are considered in application to all aggregate states of a substance, including phase transitions, and to the change in dissipative structures in open systems.Translated from Inzhenerno-Fizicheskii Zhurnal, Vol. 53, No. 5, pp. 709–716, November, 1987.     

Programmable control of column selectivity for temperature-programmed GC

A computer-driven pressure controller connected to the junction point of a series-coupled ensemble of two capillary GC columns having different stationary-phase selectivity is used to obtain on-the-fly (programmable) changes in ensemble selectivity. Changes in the junction-point pressure result in differential changes in the local carrier gas velocity in the two columns, and this results in changes in the pattern of peaks eluting from the ensemble. When used with relatively fast temperature programming (30 degrees C/min), the pattern of eluting peaks can be very sensitive to the time at which a selectivity (junction-point pressure) change is implemented. These elution pattern changes are described for a set of six PCB congeners that elute with a small range of retention times. The components are considered as a group, and changes in their elution pattern are described for a single junction-point pressure change, which is implemented at various times after sample injection. If the pressure change is implemented after the components have migrated across the junction point, the final pressure has relatively little impact on the ensemble retention pattern. Pressure changes implemented prior to the components reaching the junction can have a large effect and usually result in a pattern of peaks similar to the pattern obtained when the final pressure is used for the entire separation. For pressure changes made when the group of components is near the junction point, the observed peak pattern may be very sensitive to the time of the pressure change. The time at which the junction-point pressure change occurs is varied in 1.0-s intervals. Artifacts such as peak doubling and peak focusing or broadening are observed if a migrating band is crossing the column junction point at the time of the programmed pressure change.     

C60 and C70 HPLC retention reversal study using organic modifiers

A novel equation (Guillaume Y. C. et al. Anal. Chem. 1998, 70, 608) modeling the weak polar solute retention in reversed-phase liquid chromatography (RPLC) was applied to fullerene molecules C60 and C70. In RPLC, with an organic modifier (OM)/water mobile phase, the fullerene cluster solvation energies were calculated for OM = methanol, ethanol, propanol, butanol, and pentanol. An enthalpy-entropy compensation revealed that the type of interactions between fullerenes and the stationary phase was independent of both the fullerene and organic modifier structures. The energetics of OM and OM-water cluster exchange processes in the mobile phase were investigated in relation to the carbon atom number of the hydrophobic chain of the OM. Two linear correlations were found between the Gibbs free energy changes in the solvent exchange processes which confirmed that (i) a reversal elution order existed for C60 and C70 when methanol was changed into ethanol, propanol, butanol, pentanol and that (ii) the mobile phase was dominant in governing selectivity changes in nonpolar solutes.     

Increased water retention in polymer electrolyte membranes at elevated temperatures assisted by capillary condensation

We establish a new systematic methodology for controlling the water retention of polymer electrolyte membranes. Block copolymer membranes comprising hydrophilic phases with widths ranging from 2 to 5 nm become wetter as the temperature of the surrounding air is increased at constant relative humidity. The widths of the moist hydrophilic phases were measured by cryogenic electron microscopy experiments performed on humid membranes. Simple calculations suggest that capillary condensation is important at these length scales. The correlation between moisture content and proton conductivity of the membranes is demonstrated.     

Application of artificial neural networks for prediction of the retention indices of alkylbenzenes

Artificial neural networks (ANN) with extended delta–bar–delta (EDBD) learning algorithms were used to predict the retention indices of alkylbenzenes. The data used in this paper include 96 retention indices of 32 alkylbenzenes on three different stationary phases. Four parameters: temperature, boiling point, molar volume and the kind of stationary phase, were used as input parameters. These three stationary phases are: PEG, SE-30, SQ. The 96 group data were randomly divided into two sets: a training set (including 64 group data) and a testing set (including 32 group data). The structures of networks and the learning times were optimized. The best network structure is 4–7–1. The optimum number of learning time is about 20 000. It is shown that the maximum relative error is no more than 3%. The result illustrated that the prediction performance of ANN in the field of investigating the retention behaviors of alkylbenzenes is very satisfactory.     

Retention of ionizable compounds on HPLC. 12. The properties of liquid chromatography buffers in acetonitrile-water mobile phases that influence HPLC retention

The addition of acetonitrile to aqueous buffers to prepare RP HPLC mobile phases changes the buffer properties (pH and buffer capacity). This variation is studied for ace tate, phosphate, phthalate, citrate, and ammonia buffers in acetonitrile-water mixtures up to 60% in acetonitrile (v/v). Equations are proposed to relate pH and buffer capacity change of these buffers to the initial aqueous pH value and to the volume fraction of acetonitrile added. It is demonstrated that the pH change of the buffer depends not only on the initial aqueous pH of the buffer and on the percentage of acetonitrile added but also on the particular buffer used. The proposed equations allow an accurate prediction of this ionization for the studied buffers. Since the retention of acid/base compounds shows a strong dependence of their degree of ionization, the equations are used to predict the change in this ionization with addition of acetonitrile when the RP HPLC mobile phase is prepared. This prediction allows estimation of the retention of an acid/base compound in a particular acetonitrile-water buffered mobile phase.     

Retention indexes for temperature-programmed gas chromatography of polychlorinated biphenyls

A noninteger retention index was defined based on a series of PCB internal standards, namely congeners 8 (2,4'-dichlorobiphenyl), 31 (2,4',5-trichlorobiphenyl), 44 (2,2',3,5'-tetrachlorobiphenyl), 101 (2,2',4,5,5'-pentachlorobiphenyl), 138 (2,2',3,4,4',5'-hexachlorobiphenyl), 180 (2,2',3,4,4',5,5'-heptachlorobiphenyl), and 194 (2,2',3,3',4,4',5,5'-octachlorobiphenyl). These retention index markers are common congeners present in technical mixtures and most environmental samples, and they show a linear dependence of retention time on the number of chlorine atoms, in the temperature-programmed analysis. The index values are calculated with a single regression equation instead of the Van den Dool and Kratz equation. The retention indexes of all 209 PCBs on two commonly used columns (DB-XLB and DB-5), as well as on a supplementary column of DB-17 in capillary gas chromatography, were determined using this system. The reliability of the retention index is quite good, with the average 95% confidence limits for three measurements on each PCB being +/-0.1 index unit under the same chromatographic conditions and +/-0.4 index unit under different column head pressures. The effect of heating rate of the programmed runs on the retention index was also investigated. The inversion of the elution order of some congener pairs on the DB-XLB column for different temperature heating rates was observed. Our index values were compared with those of Castello and Testini.