On the chromatography mechanism of reversed-phase liquid chromatography

Summary An equation is derived which can describe how the retention of solutes is influenced by the composition of the mobile phase in reversed-phase liquid chromatography, the retention of solutes in alkyl bonded stationary phase regarded as the complexation between solute molecule and the active sites on the surface of the stationary phase. When the stationary phase is not fully saturated by the organic modifier, the activity of the active sites, the activity coefficient of the adsorbed solute as well as the activity coefficient of the solute in the mobile phase depend on the composition of the mobile phase. However, when the stationary phase is fully saturated, the composition of the mobile phase mainly influences the activity coefficient of the solute in the mobile phase. In addition, the selectivity of retention is discussed in terms of the derived equation.     

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.     

Retention prediction of o-phthalaldehyde amino acid derivatives in reversed-phase liquid chromatography

Summary Retention prediction of o-phthalaldehyde amino acid derivatives in reversed-phase liquid chromatography has been investigated. The retention of all derivatives could be predicted within about 10% relative error under the appropriate separation conditions in both isocratic and gradient-elution modes.     

Correlation between retention and the C18 silica surface coverage in reversed-phase liquid chromatography

Summary Members of three homologous series and five non-homologous solutes with various functional groups were chromatographed on a series of well-characterized C₁₈ reversed stationary phases with a range of methanol-water mobile phases. Measured capacity factors of solutes were correlated with the concentration of C₁₈ ligands on the stationary phase. A linear relationship with the slope depending on a solute molecular structure and the volume fraction of methanol in the mobile phase was obtained. A method for the evaluation of phase ratio is also proposed. Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Convergence of retention for small solutes in reversed-phase liquid chromatography

Summary It is shown theoretically that when the concentration of organic solvent in the mobile phase increases, or solute size decreases, log k values of small solutes in reversed-phase liquid chromatography (RPLC) will tend to have a minimum value called the convergence point. A theoretical model for evaluating the convergent coordinates of small solutes is presented by using a stoichiometric displacement model for retention (SMDR). The physical meaning of the coordinates of each kind of convergence are also elucidated. The convergence points have either two-dimensional coordinates with a common ordinate (the logarithm of the phase ratio of the column, log ) or threedimensional corrdinates with two common axes: — log and the logarithm of the molar concentration of the pure displacing agent in mobile phase, log a_D. The other axis relates to the nature of the solutes, such as carbon number of a homolog, van der Waal's surface area, hydrophobic fragment constant etc. for the latter and those and/or concentration axis for the former. The model was tested with published data and found to give a good fit.     

Detection by ion-pairing probes in reversed-phase liquid chromatography

Summary Compounds without own detector response can be detected and quantified by a UV-absorbing or fluorescent, ion-pairing probe in reversed-phase liquid-solid systems. Charged as well as uncharged samples give response by affecting the distribution of the probe between the phases. The probe is usually a hydrophobic organic ion. It is included in the mobile phase, which also contains a counter ion and an ion of the same charge as the probe, both fairly hydrophilic. The choice of properties and concentrations of the hydrophilic ions is essential for the response since too low and too high distribution of the mobile phase ions to the adsorbent will make the system insensitive. It is with UV-absorbing probes possible to get a response corresponding to a molar absorptivity of more than 3000 for ionic samples. Fluorescent probes have given 3–5 times higher sensitivity. Uncharged compounds give as a rule lower response. A combination of two UV-absorbing probes with different charge gives a considerable improvement of the response for samples with low retention. A simplified theoretical model for the detector response is suggested on the basis of detailed studies of the concentration changes in the eluted mobile phase.     

Optimizing separations in reversed-phase liquid chromatography by varying pH and solvent composition

Summary An interpretive optimization procedure in which pH can be one of the variables is presented with the emphasis on optimizing separations. When varying the pH in reversed-phase liquid chromatography the retention of ionogenic solutes will change. Thus, the selectivity between ionogenic and neutral solutes or between ionogenic solutes mutually can be optimized. However, pH also greatly affects the efficiency (plate count) and peak shape (asymmetry). Optimum selectivity (i.e. large differences in retention times) may be observed under conditions where peaks are broad and asymmetrical. Thus, it is essential to simultaneously consider retention, peak width and peak shape and their effects on separation (effective resolution) in pH-optimization studies. A procedure in which this is done is presented and applied to optimizing the separation of a synthetic mixture of selected pharmaceuticals. After initial experiments to establish the parameter space (boundaries for pH and binary methanol — water composition), twelve experiments are performed according to a 3×4 experimental design. At each loaction the retention, peak height, peak area and peak symmetry are recorded for each solute. These data are then used to build models for each of the four characteristics and for each solute. From this set of models the response surface, describing the quality of separation as a function of pH and composition, can be calculated. A variety of optimization criteria (quantifying quality of separation) can be used. The optimum corresponds to the highest point on the response surface.     

Retention prediction of phenylthiohydantoin amino acid derivatives in reversed-phase liquid chromatography

Summary The concept of retention prediction for the separation of phenylthiohydatoin-amino acid derivatives in isocratic reversed-phase liquid chromatography is described. A novel retention-solubility parameter, R, is defined, for the retention prediction strategy and the performance of this R value is evaluated by comparing measured and predicted retention data. Excellent agreement between these values were observed. It is concluded that the R value has a very high potential in describing the retention of phenylthiohydantopinamino acid derivatives withdifferent types of separation systems consisting of C-18, C-8 and phenethyl bonded stationary phases and various mobile phases.     

Retention prediction in reversed-phase liquid chromatography systems with methanol/water mobile phases containing different alkanols as additives

In an effort to gain enhancement of selectivity in reversed-phase liquid chromatography, retention was tuned in this study by introducing short and medium straight-chained-length alkanol additives (methanol (MeOH), ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol or 1-heptanol) at low concentrations in mobile phases containing MeOH as the main organic solvent. A six-parameter retention model considering simultaneously the contents of the main organic modifier and of the alcohol additive as well as of the number of alkyl chain of additive was developed by a direct combination of equations expressing separately a linear dependence of the retention upon each of these factors. The effectiveness of the above model was tested in the retention prediction of a mixture of six alkylbenzenes under isocratic conditions with mobile phases containing as an additive any member of the homologues series of alkanols (with 1-7 carbon atoms) at different low concentrations in a wide range of MeOH-water mixtures. The prediction was excellent in all cases even when the alkanol additives used in experiments for the fitting procedure are different than those used in chromatographic runs done for testing the prediction ability of the proposed model.     

Heteroscedasticity of retention factor and adequate modeling in micellar liquid chromatography

The two concepts of micelle formation (pseudo-phase and mass-action) could be the basis of retention models in micellar liquid chromatography (MLC). The separation of 4-hydroxybenzoic acid esters and seven polyaromatic hydrocarbons were performed to study the repeatability of retention factor in MLC. The full two factor experimental design was used for studying the dependence of retention factor variance on mobile phase composition (sodium dodecylsulfate, 1-butanol). The experimentally observed heteroscedasticity and perturbations after linearization were taken into account by using statistical weights obtained on the basis of errors propagation law and the modeling of retention by non-weighted and weighted least squares method was performed. The mechanistical retention models based on pseudo-phase and mass-action concepts of micelle formation were compared by fitting quality and prediction capability and high robustness of bilogarithmic dependence was observed. The significance of retention factor heteroscedasticity for retention hydrophobicity relationships was shown.     

Comprehensive two-dimensional liquid chromatography: Ion chromatography × reversed-phase liquid chromatography for separation of low-molar-mass organic acids

In the work presented here a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Ion chromatography is chosen for the first-dimension separation and reversed-phase liquid chromatography is chosen for the second-dimension separation mode. The coupling of these modes is made possible by neutralising the first-dimension effluent, containing KOH, prior to transfer to the second-dimension reversed-phase column. A test mixture of 24 low-molar-mass organic acids is used for optimisation of the system. Three food and beverage samples were analysed in order to evaluate the developed methodology, the resulting two-dimensional separation is near-orthogonal, the set-up is simple and all instrumental components are available commercially. The method proved to be robust and suitable for the analysis of wine, orange juice and yogurt.     

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.     

Capillary electrochromatography of 1-phenyl-3-methyl-5-pyrazolone derivatives of some mono-and disaccharides

Summary A packing procedure was adopted for capillary electrochromatography (CEC) that produces capillary columns with high separation efficiencies. The columns were fully packed, 50 cm long, with UV detection being performed through the packed section 30 cm from the inlet end. The CEC experiments were run at ambient pressure, with no additional pressure applied to the ends of the column. The stationary phase (octadecyl silica (ODS), 5 μm) promoted a high velocity electroosmotic flow (EOF), enabling rapid and efficient separation of a hydrocarbon test mixture. Some 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of mono- and disaccharides were baseline separated, using a 5 mM NaH₂PO₄ in 80% acetonitrile and 20% water (v/v) buffer solution. CEC shows promise for future applications in carbohydrate analysis. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

反相高效液相色谱法测定糠酸、糠醇和糠醛

提出了反相高效液相色谱法（HPLC）同时测定糠酸、糠醇和糠醛的新方法,采用C18色谱柱,流动相为V（乙腈）：V（水）=50：50,流速为1.0 mL/min;检测波长为220 nm;柱温为25℃.在4 min内可以将3种组分完全分离.糠酸、糠醇、糠醛检出限（S/N=3）分别为：0.101、0.042、0.406 mg/L.平均回收率分别为101.4%、98.5%、95.3%.相对标准偏差（RSD）分别为0.87%、0.66%、0.43%.     

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.     

Preparation and retention characteristics of different phenyl phases for reversed-phase liquid chromatography

Summary Three different phenyl phases were prepared. The amount of organic moiety bound on silica support was determined from thermogravimetric curves of the modified silica gel. The specific surface areas of gels were obtained from nitrogen sorption measurement at –196°C. The effect on separation and selectivity of the different bonded-phenyl functional groups was studied. The selectivity of the phenyl-bonded silica gels was examined and compared with octadecyl (ODS) silica in liquid chromatography. Corresponding to the high surface concentration of functional groups, the capacity factors of solutes, normalized to unit surface area of the adsorbent, k/S_BET were found to decrease in the sequence phenylmethyl>diphenyl>triphenyl. Polar solutes are retained in greater extent on the phenyl phases than on the ODS phase.     

Factors influencing retention and resolution of substituted alkylbenzenes in reversed-phase liquid chromatography

Summary The retention data of isomeric xylenes, ethyltoluenes and diethylbenzenes, and of mesitylene, benzene, toluene and ethylbenzene were obtained on a reversedphase column using methanol-water and ethanol-water mobile phases at four different temperatures. This database was used to relate the dependence of solute retention and resolution on the polarity of the mobile phase, solute dipole moment, and column temperature. The additivity of the free energy of the transfer of solute molecules or solute segments from the stationary phase to the mobile phase, was examined for the isomeric compounds. For this, the logarithm of the net retention volume was substituted for the free energy. Deviations from the additivity of free energies indicate that the separation of isomeric substituted alkylbenzenes is governed by their differential interactions with both the polar mobile phase and nonpolar stationary phase. Among the disubstituted alkylbenzenes,ortho-isomers favor the mobile phase more andpara-isomers tend to prefer the stationary phase more. Themeta-isomers are found to follow the additivity rule closely. These trends are amplified as the polarity of the mobile phase is increased indicating that these isomers are resolved better in water-rich mobile phases. These findings are substantiated by analogous results from gas-liquid chromatographic retention data, estimation of dipole moment effects, and examination of the entropic and enthalpic contributions to the net retention volume.Dedicated to Professor Leslie S. Ettre on the occasion of his 70th birthday.