Theoretical background of monolithic short layer ion-exchange chromatography for separation of charged large biomolecules or bioparticles

The retention and peak spreading in linear gradient elution of charged large biomolecules were investigated by using numerical simulations. Oligo-DNA separation by monolithic anion-exchange chromatography was chosen as a model system. The peak width and the retention were well predicted by using the parameters obtained by gradient elution experiments at different gradient slopes. As the distribution coefficient at the peak retention volume K_R decreases with increasing molecular size, the peak became sharper for larger DNAs. This is due to very large effective charge (binding site) values of large DNAs (20–60). The peak width was well correlated with K_R based on the model equation developed for linear gradient elution of proteins. It was shown that the monolithic disk is best suited for very large charged biomolecule separations at high flow velocities with shallow gradients slopes.     

Rapid Screening of Lipase Inhibitors from Ophiopogonis Radix Using High-Performance Thin Layer Chromatography by Two Step Gradient Elution Combined with Bioautographic Method

In this study, a high-performance thin layer chromatography (HPTLC) method by two step gradient elution with two mobile phases was developed for the simultaneous analysis of seven constituents in Ophiopogonis Radix. The chromatography was performed on silica gel 60 F₂₅₄ plate with dichloromethane-methanol-ethyl acetate-water (70:25:12:3, v/v/v/v) and dichloromethane-methanol (300:1, v/v) as the mobile phase for two step gradient elution. Then, the HPTLC profiles were observed after derivatization with 10% sulfuric acid in ethanol solution. The obtained HPTLC images were further analyzed by chemometric approaches and the samples could be clustered based on regions and/or growth years, which were two important factors affecting the constituents in Ophiopogonis Radix. Furthermore, five compounds including ophiopogonin D, ophiopojaponin C, ophiopogonin D’, ophiopogonin C’ and methylophiopogonanone B were screened as potential lipase inhibitors from Ophiopogonis Radix by the HPTLC-bioautographic method. The binding modes and interactions between the five compounds and lipase were further explored by molecular docking analysis. The developed HPTLC method could be used for quality control of Ophiopogonis Radix and screening of the potential lipase inhibitors.     

Determination of flavonoids,phenolic acid and polyalcohol in Butea monosperma and Hedychium coronarium by semi-preparative HPLC Photo Diode Array (PDA) Detector

The semi preparative HPLC method with PDA Detector was proposed for the determination of one phenolic acid, three flavonoids and one polyalcohol from Butea monosperma and Hedychium coronarium in gradient elution system. The influence of composition of the mobile phase concentration of the mix modifier and temperature on the separation of gallic acid, quercetin, iso-butrin, butrin and eugenol for 90 min was studied. Two different gradient programmes were used to separate these components. The lower limit of quantification of phenolic acid, flavonoids and eugenol is 0.050–0.150 μg/mL and was determined by the least square method and a good correlation was obtained for all separated components.     

Axial temperature gradient and mobile phase gradient in microcolumn high-performance liquid chromatography

The effect of axial temperature gradient (ATG) along a microcolumn on the separation performance at both isocratic and gradient elution mode was investigated. A thermostat system was designed to form an ATG along the packed column. Polycyclic aromatic hydrocarbons (PAHs) were separated on a 0.53 mm  × 150 mm i.d. 5 μm C₁₈ microcolumn, with water and acetonitrile as mobile phase. The separation results obtained at mobile phase gradient (MPG) and ATG in microcolumn HPLC were compared with the results performed at ambient conditions. Extrapolated curves of peak width at half height (wh)versus lnk showed that wh is narrower at the same retention time when ATG was applied in addition to MPG. The column efficiency was enhanced 20-30% and the resolution was slightly reduced because of reduction of selectivity at elevated temperature at ATG condition. The RSD of retention time in ATG mode was less than 2.5%.     

Relationship between human IgG structure and retention time in hydroxyapatite chromatography with sodium chloride gradient elution

Accurate prediction of the elution tendency of monoclonal antibodies in column chromatography would be beneficial for the efficient setup of purification procedures. Hydroxyapatite chromatography experiments using 37 recombinant human monoclonal antibodies were performed by sodium chloride gradient elution with 5 mM sodium phosphate to correlate the retention times with antibody structures (subclass and light‐chain isotypes). The contribution of metal affinity interactions in the interaction of antibodies with hydroxyapatite was investigated by (i) eliminating 5 mM sodium phosphate in buffers, (ii) comparing sodium chloride versus sodium phosphate gradient elutions, and (iii) using IgG₄ antibodies with a leucine→glutamate mutation. By using antibodies of different subclasses but with identical Fab regions, the elution behavior in sodium chloride elution could be classified by subclass and type of light chain. It is considered that the retention of monoclonal antibodies to hydroxyapatite is affected by the cooperation of phosphoryl cation exchange and metal affinity interactions. The contribution of the metal affinity interactions is greater in the sodium chloride gradient elution method than in the sodium phosphate gradient elution method.     

The use of mobile phase pH and column temperature to reverse the retention order of enantiomers on chiral-AGP®

Summary Enantiomeric separation of mosapride and a structurally related compound was performed using chiral chromatography and experimental design. Unique effects of mobile phase pH and column temperature made it possible to control the elution order of the enantiomers when using Chiral-AGP as the solid phase. At a low mobile phase pH (<6) the (R)-enantiomer of mosapride elutes before the (S)-form whereas the (S)-enantiomer elutes first at a high mobile phase pH (>6). By using a mobile phase pH around 6, the column temperature could also be used to control the elution order of the enantiomers of mosapride. Similar effects of mobile phase pH and column temperature were obtained for the enantiomers of a structurally related compound, a metabolite (M1). Isocratic chromatographic systems made it possible to determine enantiomeric impurities less than 0.1% in the respective enantiomer of mosapride. The enantiomers of mosapride as well as the enantiomers of M1 could easily be separated simultaneously using Chiral-AGP and a simple gradient elution. Part of this work has been presented as lectures at HPLC'96 in San Francisco USA, at AAPS-96 in Seattle USA and as a poster at HPLC'95 in Innsbruck Austria.     

Profiling the Photochemical-Induced Degradation of Rat Growth Hormone with Extreme Ultra-pressure Chromatography–Mass Spectrometry Utilizing Meter-Long Microcapillary Columns Packed with Sub-2-µm Particles

In recent years, protein therapeutics have seen increasing use in the therapeutic arena. As with traditional small molecule drug substances, one is obligated to ensure purity and stability of the various dosage forms. With these higher molecular weight therapeutics, a common approach for analytical characterization is enzymatic digestion followed by gradient elution liquid chromatography with mass spectrometry detection to create a peptide map (bottom-up protein analysis). Due to the difficulty to separate mixtures frequently encountered, there is the need for advanced chromatographic systems featuring increased resolution and/or peak capacity that can be operated in the gradient elution format. Presently, we describe an extreme ultra-pressure liquid chromatography (XUPLC) system that has been implemented as an in-house add-on to a commercial ultra-pressure chromatography system. This add-on allows operation at the 38 Kpsi range, accommodates the use of capillary columns in excess of 1 m packed with sub-2-µm particles and can be operated in the gradient elution format. To evaluate the utility of this system, rat growth hormone was used as a model protein and was exposed to light (λ 254 nm) to create a stress environment. When enzymatic digests of control and stressed protein were analyzed with the XUPLC system using MS detection, greater than 92% peptide coverage was achieved, including the identification some peptides where pre-oxidation of Met residues had occurred, as well as chemistry specifically related to the photolysis of protein disulfide linkages. When the same samples were analyzed by commercial UPLC and compared to the XUPLC results, the utility of the increased peak capacity available with the XUPLC was apparent as previously co-eluting peaks were now well resolved. In particular, one specific degradation route was identified where a pair of isobaric cis/trans diastereomerically related peptides were well resolved by XUPLC while they were unresolved by UPLC. Clearly the use of this system operating at a higher pressure regime with long capillary columns is and will be useful in continued investigations of protein stability, especially in cases where only subtle differences in the amino acid residues have occurred during degradation.     

Laguncularia racemosa Phenolics Profiling by Three-Phase Solvent System Step-Gradient Using High-Performance Countercurrent Chromatography with Off-Line Electrospray Mass-Spectrometry Detection

The detailed metabolite profiling of Laguncularia racemosa was accomplished by high-performance countercurrent chromatography (HPCCC) using the three-phase system n-hexane–tert-butyl methyl ether–acetonitrile–water 2:3:3:2 (v/v/v/v) in step-gradient elution mode. The gradient elution was adjusted to the chemical complexity of the L. racemosa ethyl acetate partition and strongly improved the polarity range of chromatography. The three-phase solvent system was chosen for the gradient to avoid equilibrium problems when changing mobile phase compositions encountered between the gradient steps. The tentative recognition of metabolites including the identification of novel ones was possible due to the off-line injection of fractions to electrospray ionization mass spectrometry (ESI-MS/MS) in the sequence of recovery. The off-line hyphenation profiling experiment of HPCCC and ESI-MS projected the preparative elution by selected single ion traces in the negative ionization mode. Co-elution effects were monitored and MS/MS fragmentation data of more than 100 substances were used for structural characterization and identification. The metabolite profile in the L. racemosa extract comprised flavonoids, hydrolysable tannins, condensed tannins and low molecular weight polyphenols.     

La séparation Lu-Yb-Tm sur échangeur de cations par l' α-ihba

The mutual separation of Lu Yb and Tm is examined with cation-exchange resin und 0.1 M αhydroxybutyric acid as eluant. When carrier amounts of ca. 1 mg of rare earths are eluted, the distribution constants are displaced and the elution peaks are asymmetrical. Quantitative separation is, however, possible at room temperature; separation requires 23 h at pH 383 but only 13 h with a pH gradient.     

Reversal of Elution Order between Enantiomers of Binaphthol on an Immobilized Polysaccharide-Based Chiral Stationary Phase

Enantioseparation of 1,1??-bi-2-naphthol (BINOL) was performed on an immobilized polysaccharide-based chiral stationary phase (CSP), Chiralpak IA, in the normal-phase mode. The effects of polar modifier in the mobile phase and column temperature on retention, enantioseparation, and elution order were investigated. An interesting reversal of elution order for BINOL was observed. When ethanol was used as a polar modifier, R-BINOL was eluted first with marginal enantioseparation. Excellent enantioseparation was obtained when ethanol was replaced by 1-propanol, and S-BINOL was eluted first, this effect being retained with 2-butanol, 1-butanol, 1-pentanol or 1-hexanol as the modifier. When isoamyl alcohol was used, reversal of elution order was again observed, i.e., R-BINOL eluted first with marginal enantioseparation, similar to the case of ethanol. When cyclohexanol and cyclopentanol were used, R-BINOL was still eluted first, but enantioseparation was as good as with 1-propanol as the modifier. This is the first report of large enantioseparation obtained in both elution orders for a given selector/selectant system. A retention model based on stoichiometric displacement theory for retention (SDT-R) was investigated to fit the chromatographic data. The reason for solvent-induced reversal of elution order was elucidated based on a derivation of the retention model. Reversal of elution order for BINOL induced by the content of isoamyl alcohol was also predicted based on the model and confirmed by experiment.     

Rapid and Complete Separation of Inorganic Polyphosphates from Monomer to 35-Mer by Hplc

Abstract

The on-line coupling system of high-performance liquid chromatography (HPLC) and flow injection analysis (FIA) was developed for the rapid separation and sensitive detection of inorganic polyphosphates. Effects of gradient elution conditions on the chromatographic behavior were studied. The column temperature as well as a chloride concentration gradient were found to be very effective for the improvement of resolution(1). To minimize analysis time and to maximize resolution, the gradient elution conditions were optimized by use of a computer-assisted retention prediction system(2). More than 35 kinds of inorganic polyphosphates could be separated completely within 200 min under the optimum conditions as shown in Fig. 1.     

Determination of phenolic compounds using high-performance liquid chromatography with Ce-Tween 20 chemiluminescence detection

A novel method for the simultaneous determination of phenolic compounds such as salicylic acid, resorcinol, phloroglucinol, p-hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, and m-nitrophenol by high-performance liquid chromatography (HPLC) coupled with chemiluminescence (CL) detection was developed. The procedure was based on the chemiluminescent enhancement by phenolic compounds of the cerium(IV)-Tween 20 system in a sulfuric acid medium. The separation was carried out with an isocratic elution or with a gradient elution using a mixture of methanol and 1.5% acetic acid. For six phenolic compounds, the detection limits (3σ) were in the range 1.40-5.02 ng/ml and the relative standard deviations (n=11) for the determination of 0.1 μg/ml compounds were in the range 1.9-2.9%. The CL reaction was well compatible with the mobile phase of HPLC, no baseline drift often occurred in HPLC-CL detection was observed with a gradient elution. The method has been successfully applied to the determination of salicylic acid and resorcinol in Dermatitis Clear Tincture and p-hydroxybenzoic acid in apple juices.     

Development of hybrid elution systems for efficient purification of stilbenoids using centrifugal partition chromatography coupled to mass spectrometry

The phytochemical study of the root extract of the stilbenoid-rich Vitis riparia × Vitis berlandieri grapevine was carried out by centrifugal partition chromatography (CPC). For this reason, we developed a new elution mode we named back-step, which allowed us to obtain cleaner fractions and a more efficient separation process when used in conjunction with a classical elution approach. Three hydroxystilbenes: (E)-resveratrol, (E)-?-viniferin and (E)-vitisin C, with greater than 90% purity were thus obtained through such process, with minimal sample handling and purification steps. Online coupling of CPC to ESI mass spectrometry was used for optimization of the separation parameters and to facilitate the characterization of the stilbenoids. This study details the first phytochemical investigation of stilbenoids from the hybrid species together with a new elution mode able to widen the range of ARIZONA biphasic systems.     

An example of quality control of fine chemical intermediates: related impurity analysis of industrial phthalic anhydride by reversed-phase high-performance liquid chromatography

A reliable reversed-phase high-performance liquid chromatographic method has been developed for analysis of related impurities in industrial phthalic anhydride (PA). Maleic acid (hydrolysis product of maleic anhydride), phthalimide, and benzoic acid were separated from phthalic acid (Pa, hydrolysis product of PA) on a C₁₈ column by gradient elution with acetonitrile and 0.1% (v/v) aqueous perchloric acid solution. This method is simple, sensitive, and accurate, and has been successfully applied to quality control of PA for industrial use.     

Effect of pore size of packing materials on molecular-weight separation by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) is an interactive polymer chromatography technique varying the column temperature during the elution in a programmed manner to control the solute retention. In the present paper, the effect of the pore size of packing materials on the molecular-weight separation of polystyrene and poly(methyl methacrylate) standard samples by TGIC was studied by using the columns (octadecyl modified silica) with different pore size (100, 300 and 1000 Å) and eluent mixture of CH₂Cl₂/CH₃CN. By rising temperature gradient, both polymers were separated by molecular weight from lower to higher. It became clear that each sample elutes out earlier as the pore size is larger. These experimental results could be explained by the theory based on the scaling concept of Gorbunov and Skvortsov.     

Simultaneous chromatographic identification of a few components in mixtures based on a correlation between their absolute or relative retention times under different conditions

A correlation was established between the absolute and relative retention parameters of various compounds under different conditions of chromatographic analysis (isothermal and temperature programming conditions in gas chromatography or isocratic and gradient elution in high-performance liquid chromatography). This correlation is described by the linear regression equations t _R (II) = = at _R (I) + b with a high degree of accuracy. This property of chromatographic retention parameters allowed us not only to recalculate the values of these parameters determined under different conditions, but also to propose an algorithm for the simultaneous identification of a few components in complex mixtures with the use of published data on retention parameters found under other conditions (even presented as the drawings of chromatograms).     

Possibilities and Limitations of Computer-Assisted Method Development in HILIC: A Case Study

In the present study, we investigated the possibilities and limitations of computer-assisted method development (CAMD) for the HILIC separation optimization of a mixture of 13 isomeric hydroxy- and aminobenzoic acids on a ZIC-HILIC column. The isocratically obtained Neue and Kuss retention parameters enabled the accurate gradient retention modeling for peaks eluting well within the gradient (mean error of 2.7 %). The prediction errors for peaks eluting at the end of the gradient could be reduced from 8.8 to 6.1 % by implementing the isocratic regime after the gradient into the expression for the gradient retention factor. The prediction of the corresponding peak widths improved significantly for certain compounds and gradient profiles using individual gradient N values for each compound compared to employing a single N value for all compounds and gradient profiles. Two gradient optimization strategies (constructing the R _s map based on individual retention modeling and predictive elution stretching and shifting, PEWS²) resulted in a reasonable separation of the challenging mixture of 13 isomeric hydroxy- and aminobenzoic acids on the ZIC-HILIC column. Overall, the optimization was limited by the steep decrease in N (dropping to the isocratic N value) and corresponding increase in peak width when increasing the gradient time. The discrimination factors d ₀ were used to assess the resolution between peaks varying widely in height. The best separation was found to be obtained via the PEWS² approach. Both the individual retention modeling and PEWS² strategies corresponded to a total instrument time less than 12 h (including equilibration). Finally, it was found that the salt concentration had a significant effect on both the retention and the peak shape of the compounds, resulting in a small “solution domain” at 10 mM. Coupled columns with higher efficiencies are suggested to improve the resolution and robustness of the separation.     

Chromatographic Fingerprint Analysis of Macrothelypteris torresiana and Simultaneous Determination of Several Main Constituents by LC

A simple and reliable identification and quality control system has been developed for monitoring a herbal plant (Macrothelypteris torresiana) based on quantitative fingerprinting analysis using liquid chromatographic method with ultra-violet detector. The herbal extract was obtained by ultrasonic-assisted extraction. The separation was performed on a Burospher-100 C₁₈ column by gradient elution with acetonitrile and aqueous phase (containing 0.5% H₃PO₄, pH 3.0) at a flow rate of 1.0 mL min^?1. Under the optimal chromatographic condition, the relative standard deviations of the retention time and the peak area were less than 0.39 and 5.51%, respectively. The intra- and inter-day precisions were ranged from 0.76 to 3.23%. Good linear behaviors over the investigated concentration ranges were obtained with the values of R ² higher than 0.999 for all analytes. The recoveries for spiked samples were in the range of 92.2–106.0%. Eleven peaks in the chromatograms of M. torresiana were identified for chromatographic fingerprint analysis. The proposed method was successfully applied to determine the contents of main constituents (protoapigenone, protoapigenin 4′-O-β-D-glucoside, apigenin 4′-O-β-D-glucoside, and apigenin) in different batches of M. torresiana. The proposed analytical procedure was proved to be a reliable and rapid method for the identification and quality control of the herbal plant M. torresiana.     

Kinetic performance limits of constant pressure versus constant flow rate gradient elution separations. Part I: theory

We report on a general theoretical assessment of the potential kinetic advantages of running LC gradient elution separations in the constant-pressure mode instead of in the customarily used constant-flow rate mode. Analytical calculations as well as numerical simulation results are presented. It is shown that, provided both modes are run with the same volume-based gradient program, the constant-pressure mode can potentially offer an identical separation selectivity (except from some small differences induced by the difference in pressure and viscous heating trajectory), but in a significantly shorter time. For a gradient running between 5 and 95% of organic modifier, the decrease in analysis time can be expected to be of the order of some 20% for both water–methanol and water–acetonitrile gradients, and only weakly depending on the value of V_G/V₀ (or equivalently t_G/t₀). Obviously, the gain will be smaller when the start and end composition lie closer to the viscosity maximum of the considered water-organic modifier system. The assumptions underlying the obtained results (no effects of pressure and temperature on the viscosity or retention coefficient) are critically reviewed, and can be inferred to only have a small effect on the general conclusions. It is also shown that, under the adopted assumptions, the kinetic plot theory also holds for operations where the flow rate varies with the time, as is the case for constant-pressure operation. Comparing both operation modes in a kinetic plot representing the maximal peak capacity versus time, it is theoretically predicted here that both modes can be expected to perform equally well in the fully C-term dominated regime (where H varies linearly with the flow rate), while the constant pressure mode is advantageous for all lower flow rates. Near the optimal flow rate, and for linear gradients running from 5 to 95% organic modifier, time gains of the order of some 20% can be expected (or 25–30% when accounting for the fact that the constant pressure mode can be run without having to leave a pressure safety margin of 5–10% as is needed in the constant flow rate mode).