Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.     

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.     

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.     

The prediction of the peak width at half height in HPLC

The prediction of the peak width at half height is an important aspect in the optimization of the chromatographic operating conditions. In this paper, a linear relationship, between the peak widths at half height and the retention values with various isocratic elution is observed. In gradient elution, however, the relationship between the peak widths at half height and the so-called invented retention values that correspond to the mobile phase composition by eluting the solute from the column end is developed. We believe that there is almost the same band width at half height inside the column (in unit of length) for different solutes. The peak width at half height in the chromatogram (in unit of time) is mainly determined by the capacity factor of the solute when it is eluted from the column end. The larger the capacity factor of a solute eluted from the column end, the more slowly will be the solute eluted from the column end and the wider will be the peak width at half height. It is possible to predict the peak width at half height in various isocratic and gradient elutions by using this linear relationship.     

Predicting the behaviour of polydisperse polymers in liquid chromatography under isocratic and gradient conditions

In this paper we describe how the existing theories to describe retention and peak width in isocratic and gradient-elution liquid chromatography can be expanded to describe the retention behaviour of natural and synthetic repetitive polymers, which feature distributions of molecules with different masses (and often different structures) rather than unambiguous molecular formulas. For polydisperse samples, it is vital that the model accommodates (isocratic) elution of sample components before the onset of a gradient, elution during the gradient, and elution after the completion of the gradient. The expanded models can readily be implemented in standard spreadsheet software, such as Excel. We have created such spreadsheets based on the conventional model for retention in reversed-phase liquid chromatography (RPLC) and on two different models for retention in normal-phase liquid chromatography. The implementation allows an easy visualization of the theoretical concept. Up to three different polymeric series can be entered, with a total of up to 100 peaks being computed and displayed in isocratic or gradient-elution chromatograms. Also visualized are "retention models" (diagrams of isocratic retention vs. composition) and "calibration curves" (retention or elution composition vs. molecular mass or degree of polymerization). The coefficients in the isocratic retention model may be correlated, as has often been observed in RPLC. It is shown that under certain conditions such a correlation corresponds to the existence of so-called critical (isocratic) conditions, at which all the members of a given polymeric series (same composition and end groups, different number of repeat units) show co-elution.     

Methacrylate monolithic capillary columns for gradient peptide separations

For the separation of peptides with gradient-elution liquid chromatography a poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) monolithic capillary column was prepared and tested. The conditional peak capacity was used as a metric for the performance of this column, which was compared with a capillary column packed with C18-modified silica particles. The retention of the peptides was found to be smaller on the BMA column than on the particulate C18 column. To obtain the same retention in isocratic elution an approximately 15% (v/v) lower acetonitrile concentration had to be used in the mobile phase. The retention window in gradient elution was correspondingly smaller with the BMA column. The relation between peak width and retention under gradient conditions was studied in detail. It was found that in shallow gradients, with gradient times of 30min and more, the peak widths of the least retained compounds are strongly increased with the BMA column. This was attributed to the fact that these compounds migrate and elute with an unfavorable high retention factor. More retained compounds are eluted later in the gradient, but with a lower effective retention factor. With shallow gradients the peak capacity of the BMA column ( approximately 90) was clearly lower than that of a conventional packed column ( approximately 150). On the other hand, with steep gradients, when components elute with a low effective retention factor, the performance of the BMA column is relatively good. With a gradient time of 15min similar peak widths and thus similar peak capacities ( approximately 75) were found for the packed and the monolithic column. Two strategies were investigated to obtain higher peak capacities with methacrylate monolithic columns. The use of lauryl methacrylate (LMA) instead of butyl methacrylate (BMA) gave an increase in retention and narrower peaks for early eluting peptides. The peak capacity of the LMA column was approximately 125 in a 60min gradient. Another approach was to use a longer BMA column which resulted in a peak capacity of approximately 135 could be obtained in 60min.     

Novel criteria for fast searching for optimal method in gradient ion chromatography: an integrated approach

In this article, an integrated approach for prediction and optimization in ion chromatography (IC) was presented. The approach provides a fast and reliable insight in the elution behavior of an IC system. The predictions are based on a mathematical model that predicts ion retentions (for both isocratic and gradient modes) by using an empirical isocratic model. Other chromatographic values significant for the optimal elution conditions (resolution, peak asymmetry) are calculated quickly and easily from the predicted retention values of characteristic points of a chromatographic peak. Every day, IC users might find this approach a suitable tool for finding optimal IC elution conditions in a given system.     

A new method of analysis of peroxydisulfate using ion chromatography and its application to the simultaneous determination of peroxydisulfate and other common inorganic ions in a peroxydisulfate matrix

A new method for the determination of peroxydisulfate using ion chromatography has been developed. Elution of peroxydisulfate was effected by isocratic elution using 200 mM NaOH at 40°C. A modification of the method using gradient elution was able to simultaneously determine other common inorganic ions (nitrate, nitrite, sulfate and chloride) down to significantly low concentrations in a peroxydisulfate matrix. The relative standard deviations (RSD) were in the range of 0.5-5%, for peak areas and <0.2% for peak retention times. The recoveries were between 95% and 120% for a concentration range of about 0.5-42 ppm. The limit of detection for peroxydisulfate ion was 0.2 ppm and for the other ions were ≤2×10(-2) ppm. The calibration curves were linear with slope and intercepts close to 1 and 0, respectively.     

Fast ion chromatography using short anion exchange columns

A fast ion chromatographic system is described which uses shorter column lengths and compares various eluent profiles in order to maximise the performance without sacrificing the chromatographic resolution. Both isocratic and gradient elution profiles were considered to find the most efficient mode of separation. The separation and determination of seven target anions (chloride, chlorate, nitrate, chromate, sulfate, thiocyanate and perchlorate) was achieved using a short (4 mm ID, 50 mm long) column packed with Dionex AS20 high-capacity anion exchange material. A hydroxide eluent was used at an initial concentration of 25 mM (at a flow-rate of 1.0 mL/min) and two performance maxima were found. The maximum efficiency occurred at a normalised gradient ramp rate of 5 mM/t₀, resulting in a peak capacity of 16, while the fastest separation (<3 min) occurred at a normalised ramp rate of 30 mM/t₀. The retention time, peak width and resolution using the different eluent profiles on varying column lengths is also compared. Further investigations in this study determined that the highest peak capacity separation under gradient conditions could be approximated using an isocratic separation. The advantage of using this novel approach to approximate the maximum efficiency separation removes the need for column re-equilibration that is required for gradient elution resulting in faster analyses and enhanced sample throughput, with benefits in particular for multidimensional chromatography.     

液相色谱梯度洗脱中的谱带压缩效应

谱带压缩效应是梯度洗脱区别于等度洗脱的重要特征。经典的范德姆特(van Deemter)理论塔板高度方程基于等度洗脱推导得到,因此不能对谱带压缩效应进行描述。在梯度洗脱中,保留因子(k)会随流动相组成(φ)的改变而发生变化,这就使得对梯度洗脱机理的研究要比等度洗脱复杂许多。该文对近10年来谱带压缩效应的研究进展,特别是溶剂强度模型(即描述ln k与φ关系的数学表达式)的非线性特征对谱带压缩因子(G)的影响进行了述评,指出为了更好地认识谱带压缩效应需要将这种非线性因素考虑在内。     

Isocratic and gradient elution chromatography: a comparison in terms of speed, retention reproducibility and quantitation

Chromatographers are cautioned to avoid gradient elution when isocratic elution will do. In this work, we compared the analytical properties of gradient and isocratic separations of a sample which can be done quite readily under isocratic conditions. We found that gradient elution gave a shorter overall analysis with similar resolution of the critical pair compared to isocratic elution without sacrificing repeatability in retention time, peak area and peak height or linearity of the calibration curve. We also obtained acceptable repeatability in peak area/height and linearity of calibrations curves for a sample that required gradient elution using a practical baseline subtraction technique. Based on these results and related work which show that columns can be reequilibrated by flushing with less than two column volumes of the initial eluent, we conclude that many of the reasons given to avoid gradient elution deserve serious reconsideration, especially for those samples which are easily separated isocratically. However, we believe isocratic elution will remain preferable when: (1) the sample contains less than 10 weakly retained components (i.e. the last peak elutes with k' < 5) or (2) the gradient baseline impedes trace analysis.     

Development of an ion chromatographic gradient retention model from isocratic elution experiments

When facing separation problems in ion chromatography, chromatographers often lack guidelines to decide a priori if isocratic elution will give enough separation in a reasonable analysis time or a gradient elution will be required. This situation may be solved by the prediction of retention in gradient elution mode by using isocratic experimental data. This work describes the development of an ion chromatographic gradient elution retention model for fluoride, chloride, nitrite, bromide, nitrate, sulfate and phosphate by using isocratic experimental data. The isocratic elution retention model was developed by applying a polynomial relation between the logarithm of the retention factor and logarithm of the concentration of competing ions; the gradient elution retention model was based on the stepwise numerical integration of the corresponding differential equation. It was shown that the developed gradient elution retention model was not significantly affected by transferring data form isocratic experiment. The root mean squared prediction error for gradient elution retention model was between 0.0863 for fluoride and 0.7027 for bromide proving a very good predictive ability of developed gradient elution retention model.     

Measuring Peak Capacity of Reversed-Phase Columns for Small Molecule Compounds Under Gradient Elution

The conventional approach to the measurement of peak capacity of reserved-phase columns under gradient elution assumes that all peaks have constant peak width, but this assumption can lead to inaccurate measurement of peak capacity. An integration approach employing a series of alkylphenones as model compounds was employed to more accurately measure the peak capacity for small molecule compounds under gradient elution. The base peak width of alkylphenones was plotted against retention time and a peak width function over retention time was established by polynomial regression. The peak capacity was then calculated by integrating the inverse of the peak width function over a gradient window. Compared to the conventional method, the integration method is not based on the assumption of equal peak width, thus providing a more accurate measurement of the peak capacity of reserved-phase columns, especially shorter ones packed with sub-2 μm particles under gradient elution.     

The Influence of Stop-Flow on Band Broadening of Peptides in Micro-Liquid Chromatography

Stop-flow techniques are occasionally needed in combinations of LC-NMR and LC-MS. During the interval when there is no flow on the column, axial diffusion of components not yet eluted can be expected to take place. In this paper the size of the band broadening which is caused by diffusion during stop-flow has been determined for two peptides on reversed-phase packed micro columns. Within a temperature range of 20–40 °C, stop-flow could be extended to 30 minutes for peptides having k values in the range of 0.7–5.1 with little increase in band width on 1.0 mm i.d. columns at isocratic conditions. Stop-flow for 6 h at 20 °C increased the peak width of bradykinin and leucine-enkephalin by 25% and 60%, respectively, depending on the secondary interactions of the peptides. The peak broadening increased with increasing temperature (from 20 to 40 °C), as expected, and the impact was significant at stop-flow times larger than 2 h. Stop-flow during gradient elution resulted in less increase in peak width than isocratic elution due to the peak compression obtained when re-entering the gradient. At 20 °C the effective diffusion coefficients of leucine-enkephalin and bradykinin were determined to 6.5 × 10⁻⁷cm ²/s and 5.5 × 10⁻⁷ cm²/s, respectively, on the packed micro column.     

Development of dual gradient column in liquid chromatography

The dual gradient column, in which both the chemical property of the stationary phase and the flow velocity in the mobile phase are heterogeneous longitudinally along the column, is developed to obtain the mobile phase gradient-like elution in an isocratic condition. Here, the step-wise dual gradient columns were prepared by connecting an inlet column (I.D. 50 microm, packed with ODS) serially to an outlet column (I.D. 100-200 microm, packed with the mixture of ODS and C1 [9:1]). The retention behavior of alkylbenzenes was able to be controlled in the dual gradient column depending on the variation in the flow velocity. Moreover, the change in retention behavior induced by the flow velocity variation for the dual gradient columns was quite different from that by the variation in organic modifier content of the mobile phase in isocratic elution for a single gradient column and can induce the similar effect with an ordinary gradient elution in a mobile phase composition.     

Relationship between isocratic and gradient retention times in the high-performance ion-exchange chromatography of proteins. Theory and experiment

Using isocratic retention parameters, the gradient elution retention time for several proteins has been calculated. The gradient retention time calculation is based on fitting the isocratic retention data to an equation of the form: log k' = m log (1/[Ca2+]) + log K and on applying well-established principles of gradient elution. A good correlation between the observed and calculated retention times for several test proteins was obtained at various total gradient times and column flow-rates. Conversely, isocratic retention parameters characterizing protein retention can be calculated from gradient elution retention data. However, even with retention data of high quality, small errors are amplified by the log-log nature of the ion-exchange isocratic retention model employed. Based on the close correlation between predicted and observed gradient retention times, no evidence for protein denaturation resulting from immobilization of the protein at high initial k' values at or near the column inlet was observed.     

Theory and applications of a novel ion exchange chromatographic technology using controlled pH gradients for separating proteins on anionic and cationic stationary phases

pISep is a major new advance in low ionic strength ion exchange chromatography. It enables the formation of externally controlled pH gradients over the very broad pH range from 2 to 12. The gradients can be generated on either cationic or anionic exchangers over arbitrary pH ranges wherein the stationary phases remain totally charged. Associated pISep software makes possible the calculation of either linear, nonlinear or combined, multi-step, multi-slope pH gradients. These highly reproducible pH gradients, while separating proteins and glycoproteins in the order of their electrophoretic pIs, provide superior chromatographic resolution compared to salt. This paper also presents a statistical mechanical model for protein binding to ion exchange stationary phases enhancing the electrostatic interaction theory for the general dependence of retention factor k, on both salt and pH simultaneously. It is shown that the retention factors computed from short time isocratic salt elution data of a model protein can be used to accurately predict its salt elution concentration in varying slope salt elution gradients formed at varying isocratic pH as well as the pH at which it will be eluted from an anionic exchange column by a pISep pH gradient in the absence of salt.     

Desorption of plasmid DNA from anion exchangers: Salt concentration at elution is independent of plasmid size and load

Detailed studies on the sorption behavior of plasmids on anion exchangers are rare compared to proteins. In this study, we systematically compare the elution behavior of plasmid DNA on three common anion exchange resins using linear gradient and isocratic elution experiments. Two plasmids of different lengths, 8 and 20 kbp, were studied and their elution characteristics were compared to a green fluorescent protein. Using established methods for determining retention characteristics of biomolecules in ion exchange chromatography lead to remarkable results. In contrast to the green fluorescent protein, plasmid DNA consistently elutes at one characteristic salt concentration in linear gradient elution. This salt concentration was the same independent of plasmid size but differed slightly for different resins. The behavior is consistent also at preparative loadings of plasmid DNA. Thus, only a single linear gradient elution experiment is sufficient to design elution in a process scale capture step. At isocratic elution conditions, plasmid DNA elutes only above this characteristic concentration. Even at slightly lower concentrations most plasmids remain tightly bound. We hypothesize, that the desorption is accompanied by a conformational change leading to a reduced number of available negative charges for binding. This explanation is supported by structural analysis before and after elution.     

High-performance liquid chromatography of sialooligosaccharides and gangliosides

Glycans were cleaved from gangliosides and separated by high-performance liquid chromatography (HPLC). The columns were packed with bonded stationary phases made of microparticulate, macroporous silica with serotonin, phenylpropanolamine or tryptamine as the biogenic amine ligate. The ganglioside oligosaccharides were eluted in the order of increasing number of sialic acid residues in the molecule and their retention decreased with the ionic strength of the mobile phase. Best selectivity was obtained in the pH range from 3.0 to 4.0. The two major sialic acids, N-acetylneuraminic and N-glycolylneuraminic acids, were separated by lectin affinity chromatography using an HPLC column packed with silica-bound wheat germ agglutinin and 10 mM phosphate buffer, pH 4.0, as the eluent. Throughout this study, isocratic elution was used and the column effluent was monitored at 195 nm.