Prediction of peptides retention behavior in reversed-phase liquid chromatography based on their hydrophobicity

Hydrophobicity is an important physicochemical property of peptides and proteins. It is responsible for their conformational changes, stability, as well as various chemical intramolecular and intermolecular interactions. Enormous efforts have been invested to study the extent of hydrophobicity and how it could influence various biological processes, in addition to its crucial role in the separation and purification endeavor as well. Here, we have reviewed various studies that were carried out to determine the hydrophobicity starting from (i) simple amino acids solubility behavior, (ii) experimental approach that was undertaken in the reversed-phase liquid chromatography mode, and ending with (iii) some examples of more advanced computational and machine learning models.     

反相液相色谱中溶质保留与溶剂化结构参数间的定量关系

本文研究了等度条件下反相液相色谱中溶质保留与溶剂化结构参数间的定量关系;考察了反相液相色谱保留值变化规律式lnk'=a+cC~b中参数a.c 与溶剂化结构参数间的定量关系,结果表明能较好地预测等度和不同冲洗剂组成下溶质的保留值,将乙腈-水和甲醇-水冲洗剂下的a.c值定量关联,表明在考虑溶质的氢键作用参数后,其相关性有所提高.     

Relationship between the retention characteristics on an alumina column and physico-chemical parameters of some environmental pollutants

The retention behavior of 16 environmental pollutants was studied on alumina and porous graphitized carbon (PGC) columns using n-hexane as eluent. The relationship between the logarithm of the capacity factors determined on the alumina column and the physico-chemical characteristics of the solutes was elucidated by principal component analysis (PCA) followed by two-dimensional nonlinear mapping. The 12 original variables can be reduced to four with only a 10% loss of information. The logarithm of the capacity factor formed a cluster with the hydrogen donor and acceptor properties of the solutes and their Taft's constant on the two-dimensional nonlinear map of PC loadings indicating that both steric and electronic parameters play a considerable role in the retention mechanism on alumina support. Alcohols, aromatics and chlorinated alkanes formed separate clusters on the two-dimensional nonlinear map of PC variables suggesting that their retention mechanism may be different on the alumina column. Solutes were not retained on the PGC surface proving that under normal-phase conditions the retention capacity of alumina can be higher than that of PGC.     

Thermodynamic studies of pressure-induced retention of peptides in reversed-phase liquid chromatography

The pressure-induced retention of peptides on reversed-phase HPLC was studied by systematically changing organic solvent composition and temperature at both low (19 bar) and high (318 bar) pressures using a homologous series of hydrophobic poly-L-phenylalanine (n = 2-7) as the model compound. Based on van' t Hoff plots under different organic solvent compositions and pressures, the enthalpy change for the solute (deltaH) was determined. Moreover, both the enthalpy and entropy change for each phenylalanine residue (deltadeltaH and deltadeltaS), which corresponds to solute retention on a microenvironment along the depth of C18 chain, were also calculated by direct subtractions. Results indicate that under acetonitrile (ACN) compositions above 35%, the pressure caused deltadeltaS value to change from a negative to a positive value and both deltaH and deltadeltaH to change from a negative to a less negative value, all leading to a thermodynamic state closer to those under 35% acetonitrile composition. This implies that the pressure-induced retention observed in this study was an entropy-favored but enthalpy-unfavored process and was explained by pressure-induced desorption of solvent molecules that were associated with the stationary phase or with the peptide solute. Under 35% acetonitrile composition, however, it was found that neither deltadeltaH nor deltadeltaS value was significantly changed by the pressure. Whereas, both deltaH value and the intercept of van't Hoff plots under 35% acetonitrile composition were increased by pressure. This indicates that under low organic solvent composition, 35%, most of the acetonitrile molecules adsorbed on the surface of the stationary phase and only little solvent molecules were dissolved in the bulk stationary phase where the phenylalanine residues were partitioned. This study has provided new thermodynamic insights to the pressure-induced retention for peptides and proteins.     

Correlation between the chromatographic retention and physicochemical properties of azoles

The chromatographic behavior of previously unknown nitrogen-containing heterocyclic compounds was studied by reversed-phase high-performance liquid chromatography. The main relationships in the retention of the compounds with variation of the acetonitrile content in the mobile phase were examined. One-and multiparameter correlations between the logarithm of the retention factor and quantities characterizing physicochemical properties of the compounds were constructed.     

The correlation between molecular polarizability of PAHs and their retention data on various stationary phases in reversed-phase HPLC

Summary The equation for the potential energy of interactions established for gas-liquid chromatography has been confirmed in reversed phase liquid chromatography. Equations derived for molecular polarizabilities of PAHs and their retention data have very high correlation coefficients. The results indicate that the inductive effect between solute and stationary phase is the main one and the dispersive effect is very small compared with the inductive effect but its contribution increases with the carbon chain length of the stationary phase.     

Effect of modifying alumina desiccants with sulfuric acid on their physicochemical properties

In the production of alumina desiccants by extrusion, the introduction of sulfuric acid at the stage of preparing a mouldable paste based on hydroxides containing bayerite or pseudoboehmite increases the sorption capacity of the product. This effect is most pronounced for the pseudoboehmite-based materials. The dynamic capacity of these desiccants increases to the level characteristic of the bayerite-containing hydroxide (>5 g H₂O/100 cm³) for a dew point of ?40°C and a contact time of 1.5 s), and their static capacity exceeds this value (increasing from 21.13 to 23.1 g H₂O/100 cm³). This procedure changes the phase composition and textural characteristics of the pseudoboehmite-based desiccants and increases Brønsted acidity and generates strong Lewis acid sites on the surface of all oxides. The dynamic capacity of desiccants with similar textural characteristics depends on the acid-base properties of their surface.     

Evaluation of octadecyl-bonded alumina for separations of proteins and peptides by reversed-phase high-performance liquid chromatography

Summary A recently-developed octadecyl-bonded alumina (ODA) stationary phase was evaluated for the separation of peptides and proteins by reversed phase high performance liquid chromatography. Using standard water-acetonitrile mobile phase gradients containing 0.1 % trifluoroacetic acid, the average peak capacity obtained for the separation of a mixture of ribonuclease a, cytochrome c, lysozyme and carbonic anhydrase on an ODA column are similar to that obtained on a widely used octadecylsilane (ODS) column. However, overall chromatographic resolution of the components of this mixture on ODA is inferior to that obtained on ODS. Cytochrome c peak areas were found to be 50% smaller on the ODA column than on ODS. On the other hand, both peak capacities and resolutions of octapeptide mixtures were found to be generally superior on the ODA column, and peak areas for a representative octapeptide were found to be virtually identical for both ODA and ODS columns. The differences in the results obtained on the ODA and ODS columns for these separations are attributed to the smaller pore size and unique fused-microplatelet shape of the ODA particles. Comparisons of the separations of the tryptic digest of cytochrome c on the ODS and ODA columns demonstrate that the ODA phase is potentially as useful as ODS for peptide mapping applications.     

Study on the relationship between retention behavior and molecular structure parameters of substituted benzene derivatives in RPLC

Summary A quantitative structure-retention model derived from the concept of hydrophobic substituent constant () and the total solubility parameter (_T) has been established. It has been used to predict successfully the retention behavior of a number of solutes on various columns in several different eluents. The experimental results indicate that the model can be employed for the prediction of retention of substituted benzenes in a large volume fraction range of mobile phase. In addition, the physical meaning of the sign and the influence of the coefficients of the model on the retention process had been discussed together with the dependence of these coefficients on the composition of mobile phase.     

Correlations of the physicochemical parameters of azaindoles with their reactions

Using the AM1 semiempirical quantum method the enthalpies of formation, ionization energies, electron affinities, energy differences between highest occupied and lowest unoccupied orbitals, atomic charges, bond orders, and dipole moments have been calculated for 4-, 5-, 6-, and 7-azaindoles. A correlation has been built up between the calculated physicochemical parameters and the Hammett para-substituent and inductive constants. The 1H to 7H proton transfer in 7-azaindole has been quantitatively described. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 7, pp. 1062–1072, July, 2006.     

Influence of different N- and O-linked carbohydrates on the retention times of synthetic peptides in reversed-phase high-performance liquid chromatography.

Glycopeptides consisting of 6-19 amino acid residues and different mono- and disaccharides attached to single asparagine and serine residues were synthesized on solid-phase and were characterized by reversed-phase high-performance liquid chromatography and circular dichroism. It was shown that the decreased retention times due to glycosylation could be correlated with the increasing length of the sugar moiety. Phosphorylation of the same sequences reduced the retention times 1.6 times more than glycosylation with monosaccharides did. The binding to the column was dependent on the structure of the disaccharide when derivatized and glycosylated asparagine, the building block of N-glycopeptide syntheses was studied. However, this structural dependence of the elution times disappeared in the final glycopeptides. Although both glycosylation and phosphorylation resulted in altered secondary structure of the peptide backbone, it appears that the retention times reflect the increased hydrophilicity more strongly than induced conformational orientation on the surface of the bonded phase.     

Study on the retention behaviour of low-molar-mass polystyrenes and polyesters in reversed-phase liquid chromatography by evaluation of thermodynamic parameters

Polymers can be characterized under sorption conditions, to obtain information on molar mass and chemical composition. In order to get a better understanding of their retention behaviour under such conditions, the evaluation of thermodynamic parameters obtained from van't Hoff analyses on low-molar-mass polystyrenes (PS) and polyesters (PE) in various THF–water mixtures on a C₁₈ column is described in this study. Linear van't Hoff behaviour was observed in almost all cases. Negative values for both ΔH and ΔS were found for both PS and PE oligomers, which increase with increasing %THF. For ΔS this is explained from multi-site attachment effects. For PS, the non-linear relations between ΔH and ΔS, and degree of polymerization (p) could be properly described by the Stockmayer–Fixman equation. Although less clear, similar trends were found for PE. For PS, evidence for penetration effects of oligomer chains into the bonded chains was obtained. Martin plots for both PS and PE were shown to be non-linear in all investigated eluent compositions. The extent of non-linearity is suggested to depend on the conformation of a polymer in solution. No distinct enthalpy–entropy-compensation temperature (EECT) independent of p was found for PS, thus confirming the findings of an earlier study in which no exact molar mass independence was found under critical conditions. Further evaluation of EECT for PS oligomers revealed a retention mechanism independent of the binary eluent composition. This indicates that conclusions from this study can also be used for a qualitative understanding of sorption mechanisms in the gradient elution mode. Finally, for PS it was shown that ΔG equals zero under critical conditions, thus confirming theoretical predictions.     

Structure and retention of 2,4-dihydroxythiobenzanilides in a reversed-phase system

The effect of substitution of the N-amide system of 2,4-dihydroxythiobenzanilides on retention in a reversed-phase HPTLC system using methanol as an organic modifier was investigated. The linear relationship between R_M and the volume fraction of organic solvent for all 60 tested compounds was obtained. These relationships allowed determination of the hydrophobicity indices, R_Mw, of these compounds using the extrapolation method. On the basis of analytical data obtained from analysis of UV–Vis and ¹H NMR spectra the effect of substitution on the charge distribution in the amide system and the effect of this distribution on phase separation in relation to theoretical values is discussed.