High-performance liquid chromatography of amino acid peptides and proteins

Summary The retention behaviour of seven globular proteins ranging in molecular weight from 12,000 to 69,000 was investigated using Mono-Q anion-exchange resin as the stationary phase and sodium chloride as the displacer salt. In particular the influence of changes in ionic strength and mobile phase pH on the isocratic retention properties was assessed. Several proteins were found to have significant retention when the pH of the mobile phase was below the reported pl values of the proteins. This behaviour results from the non-uniform charge distribution on the protein surface, which allows interaction with the charged stationary phase even though the protein net charge is equal to or greater than zero. The influence of pH and ionic strength on experimentally observed bandwidths was also investigated. The dependence of the effective reduced plate height on solute capacity factor was found to vary significantly with the mobile phase pH, a behaviour consistent with the interplay of complex multisite binding kinetics. These results provide a basis for further detailed investigations into the mechanism of interaction of proteins not only with charged surfaces associated with adsorptive chromatographic media but also with other macromolecules. For Part LXXXII, see ref. [27].     

High-performance liquid chromatography of amino acids, peptides and proteins CXXV. Molecular dynamics simulation of n-butyl chains chemically bonded to silica-based reversed-phase high-performance liquid chromatography sorbents

The molecular dynamics method has been applied to investigate the conformations of n-butyl ligands immobilised onto an amorphous silica surface analogous to those utilised with silica-based RP-HPLC sorbents. Three systems were constructed which corresponded to ligand densities of 1.64, 2.67 and 3.69 μmol/m². A number of parameters related to the structure of the sorbent materials were derived in order to characterise the molecular properties of each system. These parameters included the hydrocarbon layer thickness, the frequency of gauche conformations, the distance distribution for carbon atoms and the diffusion coefficients of individual atoms in the n-butyl ligands. From these properties, the positions of chains with respect to the surface as well as their mobility were estimated. It was found that at higher densities, the n-butyl chains are predominantly perpendicular to the surface while at low density they are highly tilted or lying almost parallel to the surface. The degree of ligand flexibility decreased with increasing surface density. Mobility of individual carbon atoms as well as chain disorder increased with distance from the surface for all ligand densities. The simulated properties of n-butyl chains immobilised to a silica surface correlated well with results obtained by Fourier transform IR and ¹³C-cross polarisation magic angle spinning NMR experimental methods and statistical predictions of the behaviour of immobilised chains.     

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXIV. Application of derivative spectroscopy to the study of column residency effects in the reversed-phase and size-exclusion liquid chromatographic separation of proteins

The influence of column residency times on the reversed-phase gradient elution behaviour of human and bovine growth hormones has been investigated using on-line photodiode array spectroscopic detection. Stationary phase induced effects on protein conformation were monitored by changes in the maxima to minima ratio of the second derivative spectrum of the eluted protein. Significant changes in the second derivative ratio of the unmodified and the fully reduced and alkylated protein were observed following long incubation times, i.e. t(dwell) greater than 15 min, at the stationary phase surface in the presence of 0.1% trifluoroacetic acid before elution with a 0-75% aqueous acetonitrile gradient. The application of multi-wavelength detection in the study of equilibrium unfolding of growth hormones in urea by size-exclusion chromatography was also investigated. On-line photodiode array instrumentation and derivative spectra rationing was employed to monitor tertiary and quaternary structural changes associated with protein denaturation during a chromatographic separation. These studies clearly demonstrate the powerful detection capabilities of such instrumental approaches for the on-line evaluation of both stationary phase surface and/or mobile phase mediated changes in protein conformation.     

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.     

High-speed assay of amino acids using reversed-phase liquid chromatography

A procedure is described by which 20 commonly occurring amino acids may be separated by reversed-phase liquid chromatography at room temperature in a total time, including derivatisation with o-phthalaldehyde/2-mercaptoethanol reagent, of approximately 15 minutes. Further, an increase in column temperature enables increased resolution of certain amino acids to be obtained with small but acceptable losses in fluorescence intensity. The only major drawback to the method is that the derivatisation reagent does not react with proline or hydroxyproline.     

High-performance liquid chromatography of amino acids, peptides and proteins. LXXXV. Evaluation of the use of hydrophobicity coefficients for the prediction of peptide elution profiles

The gradient elution behaviour of five synthetic decapeptide analogues has been investigated using an octadecylsilica stationary phase and trifluoroacetic acid-water-acetonitrile mobile phases. The influence of gradient time and flow-rate on the relative retentions and bandwidths of these peptides was assessed using quantitative expressions derived from linear solvent strength theory and general plate height theory. Linear relationships between logarithmic median capacity factors, log k, and the mole fraction of organic solvent modifier, phi, were observed over the experimental range of conditions used. The slopes of these plots were different for all peptides, which indicates that divergences will occur in the prediction of peptide retention times due to conformation dependent changes in hydrophobic contact area occupancy at the stationary phase surface. However, the differences in S values (tangent to the curve obtained in a plot of log k versus phi) for these peptides were not substantial enough to seriously affect the prediction of peptide retention times at one gradient slope from those observed at another. In addition, significant differences existed between experimental and theoretical peak capacity data of these peptide analogues of similar molecular weight and overall polarity, particularly at lower flow-rates or longer residence times. These results once again demonstrate that additional diffusional and interactive processes occur during the reversed-phase separation of peptides and proteins which are not yet adequately formalized by current chromatographic theory.     

Applications of hydrophilic interaction chromatography to amino acids,peptides, and proteins

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed‐phase liquid chromatography, in a two‐dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited.     

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C4 with trifluoroacetic acid (TFA)-acetonitrile. The separation patterns for recombinant derived interleukin-1 beta (IL-1 beta) on the C4 column eluted with TFA-acetonitrile and the DVB column eluted with acetic acid-acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1 beta preparation. Neither eluent had any harmful effect on the biological activity of IL-1 beta isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol or water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80-90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without organic modifiers were found to be similar and comparable to those obtained on a silica C4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA-acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was surprisingly pure (as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.     

High-performance liquid chromatography of membrane proteins

Various modes of high-performance liquid chromatography, gel filtration, ion-exchange chromatography, hydrophobic interaction chromatography, reversed-phase chromatography and metal chelate affinity chromatography, were investigated for the separation of membrane proteins. All were found applicable to membrane proteins, although the usefulness of each mode differed. For satisfactory results it was important to select appropriate elution conditions. The type and concentration of detergent was of special importance. The effects of other conditions, flow-rate, gradient steepness, type of buffer and salt, eluent pH, etc., were similar to those observed for soluble proteins.     

Retention prediction of small peptides in reversed-phase liquid chromatography

Summary Retention prediction of small peptides (up to four residues) in reversed-phase liquid chromatography has been investigated, considering the contributions of side chains in each position to the peptide retention. In isocratic elution the retention of peptides could be predicted within about 8% relative error.     

Retention behaviour of polystyrene oligomers in reversed-phase liquid chromatography

Reversed-phase liquid chromatography (RPLC) was employed to investigate the behaviour of low-molecular-mass polystyrene oligomers with three different end groups, n-butyl, sec-butyl, and tert.-butyl polystyrenes. Exothermodynamic retention studies on the polystyrene oligomers were carried out using a C18 stationary phase column and 100% methanol mobile phase over the temperature range 15 to 60 degrees C. The resulting van't Hoff plots were linear over the entire temperature range for all three end group polystyrenes. Enthalpy-entropy compensation (EEC) showed a linear compensation for the higher-order oligomers, but was non-linear for the lower-order oligomers, indicating a change in the mechanism of retention. Differences in the extent of retention for each of the three end groups were also apparent. The ramifications of these differences are discussed.     

Behavior of large nucleic acids in reversed-phase high-performance liquid chromatography.

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Analysis shows that the retention time depends not only on the chain length but also on the base composition and the secondary structure of the molecule. A model is proposed to interpret their behavior. This model, called "multiple-point interaction theory" is based on the observation that macromolecules are flexible and very large compared to the hydrophobic phase (octadecylsilane) of the column. It explains the behavior of large nucleic acids in terms of an equilibrium of the macromolecule between the two phases through a multiple-point attachment to the chromatographic matrix, the parameters of the equilibrium being both the hydrophobicity of the base and the number of attachment points. This model fits the experimental data and can be applied to all types of flexible macromolecules, especially proteins and nucleic acids, when they are chromatographed on reversed-phase columns. The model is used to explain the separation of nucleic acids of importance in molecular biology.     

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.     

Separation of racemic 2,4-dinitrophenyl amino acids on zirconia-immobilized quinine carbamate in reversed-phase liquid chromatography

Zirconia is known to be one of the best chromatographic support materials due to its excellent chemical, thermal, and mechanical stability. A quinine carbamate-coated zirconia was prepared as a chiral stationary phase for separation of enantiomers of DNP-amino acids in reversed-phase liquid chromatography. Retention and enantioselectivity of this phase were compared to those for quinine carbamate bonded onto silica. Most amino acids studied were separated on the quinine carbamate-zirconia CSP although retention was longer and chiral selectivity was somewhat lower than on the corresponding silica CSP. Increased retention and decreased selectivity are probably due to strong non-enantioselective Lewis acid-base interactions between the amino acid molecule and the residual Lewis acid sites on the zirconia surface.     

Correlation between hydrophobicity of amino acids and retention data in reversed-phase liquid chromatography with micellar eluents

Summary Hydrophobic character is usually expressed in terms of the partition coefficient in 1-octanol-water (log P_O/W). However, measurement of this coefficient is often problematic. Retention in micellar liquid chromatography is mainly due to hydrophobic interactions and can also be used as an index of hydrophobicity. A hydrophobicity scale was established with retention data foro-phthalaldehyde (OPA)-N-acetyl-L-cysteine (NAC) amino acid derivatives, using the glycine derivative as reference. Since the OPA-NAC derivatives only differ in the nature of R₁ in the amino acid (R₁CH(COOH)NH₂), in the absence of electrostatic interactions the hydrophobic character of the substituent was responsible for retention. Linear relationships were obtained between log of the ratiok′ of amino acid derivatives:k′ of the glycine derivative for a given mobile phase, and logP _O/W for the R₁ substituent. Good correlations were also found for phenylthiohydantoin amino acid derivatives.