Peak spreading in linear gradient elution chromatography with a thin monolithic disk

The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-μm porous bead IEC column (Resource Q, 6.4mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position K(R). HETP values were constant (ca. 0.003-0.005 cm) when K(R)>5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50-100 plates for the 3mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume=0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.     

Binding site and elution behavior of DNA and other large biomolecules in monolithic anion-exchange chromatography

Our previous study has shown that there is a good correlation between the number of charges of DNA (from trimer to 50-mer) and the number of binding sites B in electrostatic interaction chromatography (ion-exchange chromatography, IEC). It was also found that high salt (NaCl) concentration is needed to elute large DNAs (>0.6 M). In this paper we further performed experiments with large DNAs (up to 95-mer polyT and polyA) and charged liposome particles of different sizes (ca. 30, 50 and 100 nm) with a monolithic anion-exchange disk in order to understand the binding and elution mechanism of very large charged biomolecules or particles. The peak salt (NaCl) concentration increased with increasing DNA length. However, above 50-mer DNAs the value did not increase significantly with DNA length (ca. 0.65–0.70 M). For liposome particles of different sizes the peak salt concentration (ca. 0.62 M) was similar and slightly lower than that for large DNAs (ca. 0.65–0.70 M). The binding site values (ca. 25–30) are smaller than those for large DNAs. When arginine was used as a mobile phase modulator, the elution position of polyA and polyT became very close whereas in NaCl gradient elution polyT appeared after polyA eluted. This was mainly due to suppression of hydrophobic interaction by arginine.     

Theoretical background of short chromatographic layers. Optimization of gradient elution in short columns

Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.     

Computational method for modeling of gradient separation in ion-exchange chromatography

A model for the simulation of the gradient separation in ion-exchange chromatography is presented. It is based on discontinuous plate model and simulates the distribution of analytes in the ion-exchange column during the separation process. It enables calculations of chromatograms for the analytes with integer and non-integer effective charges under complex gradient profiles. Equilibrium concentrations of all analytes are calculated using the same mathematical equations and expressions regardless of the effective charge on the analyte. The main parameters required for the simulations have to be determined under isocratic elution. The suitability of the model was tested with different types of gradients. A comparison of retention times and chromatograms shows that reliable predictions for all tested gradients are achieved. The observed average of the absolute values of the relative errors of the retention times obtained for any analyte in the present study from the calculated chromatograms is below 4%, while the average error considering all analytes in the study is below 2%.     

Preparative separation of bioactive constitutes from Zanthoxylum planispinum using linear gradient counter‐current chromatography

Counter‐current chromatography is a chromatographic technique with a support‐free liquid stationary phase. In the present study, a successful application of linear gradient counter‐current chromatographic method for preparative isolation of bioactive components from the crude ethanol extract of Zanthoxylum planispinum was presented. The application of n‐hexane/ethyl acetate/methanol/water quaternary solvents, in terms of “HEMWat” or “Arizona” solvent families, in gradient elution mode was evaluated. Results indicated that slightly proportional changes of biphasic liquid systems provided the possibility of gradient elution in counter‐current chromatography, maintaining stationary phase retention in the column. With the selected quaternary solvent systems composed of n‐hexane/ethyl acetate/methanol/water (2:1:2:1 and 3:2:3:2, v/v), and optimized gradient programs, in total seven fractions were separated in 4.5 h. Most of the purified compounds could be obtained at the milligram level with over 80% purity. The present study indicated that the linear gradient counter‐current chromatographic approach possessed unique advantages in terms of separation efficiency, exhibiting great potential for the comprehensive separation of complex natural extracts.     

Control of column influence on the wide range pH gradient in ion-exchange chromatography

Summary A method for optimization of the mobile phase composition in ion exchange chromatography with a wide range external pH gradient (approx. over 5 pH units) has been formulated. It is shown that programming of various wide range pH profiles which are not impaired by buffer interactions of the mobile phase with sorbent is possible. Utilization of the wide range external pH gradient in cation-exchange chromatography of bovine haemoglobin is also demonstrated.     

梯度液相色谱中梯度曲线变形对色谱峰宽的影响

在梯度液相色谱中,溶剂混合以及轴向扩散等因素会使梯度曲线发生变形,而这在阶梯梯度以及高斜率的线性梯度中表现得尤为明显。本文探讨了这种梯度曲线变形对色谱峰宽的影响。首先以C18色谱柱为固定相,甲醇-水为流动相,联苯和苯乙酮为样品,测得不同线性梯度和阶梯梯度条件下的色谱峰。然后以205 nm为检测波长,记录相应条件下未接色谱柱时甲醇的响应值,得到柱入口处的梯度曲线。接着根据所设定的梯度条件以及柱入口处测得的梯度曲线,分别计算相应情形中色谱峰宽的理论值,将其与实验值进行了比较。研究结果表明,梯度曲线的变形会对色谱峰宽产生影响。当将这种影响考虑在内后,理论值与实验值更为吻合。     

Polymer-based monolithic microcolumns for hydrophobic interaction chromatography of proteins

Monolithic capillary columns for hydrophobic interaction chromatography (HIC) have been prepared by thermally initiated, single-step in situ polymerization of mixtures of monovinyl monomers including butyl methacrylate and/or 2-hydroxyethyl methacrylate, with a divinyl crosslinker glycerol dimethacrylate or 1,4-butanediol dimethacrylate using two different porogen systems. Two porogenic solvent mixtures were used; one "hydrophilic", consisting of water, butanediol, and propanol, and one "hydrophobic," comprising dodecanol and cyclohexanol. The porous structures of the monoliths were characterized and their performance was demonstrated with a separation of a mixture of myoglobin, ribonuclease A, and lysozyme under conditions typical of HIC.     

Capacity factors and peak widths in the gradient elution reversed phase separation of high molar mass polystyrenes

Summary A simplex method for determining the constantsS andk _o from the equation lnk’=lnk _o−S ϕ (was developed and applied to the reversed phase separation of high molar mass polystyrenes using gradients of any curvature. Experimental retention times described using the equation had a standard deviation of 1.1%. The inclusion of a quadratic term in the equation was found to be unwarranted. BothS and lnk _o varied linearly with ln molar mass. The logarithm of peak width of an eluting polystyrene peak was found to be a linear function of the mobile phase composition at elution. The slope was equal toS.     

Temperature-responsive chromatography for the separation of biomolecules

Temperature-responsive chromatography for the separation of biomolecules utilizing poly(N-isopropylacrylamide) (PNIPAAm) and its copolymer-modified stationary phase is performed with an aqueous mobile phase without using organic solvent. The surface properties and function of the stationary phase are controlled by external temperature changes without changing the mobile-phase composition. This analytical system is based on nonspecific adsorption by the reversible transition of a hydrophilic-hydrophobic PNIPAAm-grafted surface. The driving force for retention is hydrophobic interaction between the solute molecules and the hydrophobized polymer chains on the stationary phase surface. The separation of the biomolecules, such as nucleotides and proteins was achieved by a dual temperature- and pH-responsive chromatography system. The electrostatic and hydrophobic interactions could be modulated simultaneously with the temperature in an aqueous mobile phase, thus the separation system would have potential applications in the separation of biomolecules. Additionally, chromatographic matrices prepared by a surface-initiated atom transfer radical polymerization (ATRP) exhibit a strong interaction with analytes, because the polymerization procedure forms a densely packed polymer, called a polymer brush, on the surfaces. The copolymer brush grafted surfaces prepared by ATRP was an effective tool for separating basic biomolecules by modulating the electrostatic and hydrophobic interactions. Applications of thermally responsive columns for the separations of biomolecules are reviewed here.     

Convective interaction media short monolithic columns: enabling chromatographic supports for the separation and purification of large biomolecules

New therapeutics that are being developed rely more and more on large and complex biomacromolecules like proteins, DNA, and viral particles. Manufacturing processes are being redesigned and optimized both upstream and downstream to cope with the ever-increasing demand for the above target molecules. In downstream processing, LC still represents the most powerful technique for achieving high yield and high purities of these molecules. In most cases, however, the separation technology relies on conventional particle-based technology, which has been optimized for the purification of smaller molecules. New technologies are, therefore, needed in order to push the downstream processing ahead and into the direction that will provide robust, productive, and easy to implement methods for the production of novel therapeutics. New technologies include the renaissance of membranes, various improvements of existing technologies, but also the introduction of a novel concept--the continuous bed or monolithic stationary phases. Among different introduced products, Convective Interaction Media short monolithic columns (SMC) that are based on methacrylate monoliths exhibit some interesting features that make them attractive for these tasks. SMC can be initially used for fast method development on the laboratory scale and subsequently efficiently transferred to preparative and even more importantly to industrial scale. A brief historical overview of methacrylate monoliths is presented, followed by a short presentation of theoretical considerations that had led to the development of SMC. The design of these columns, as well as their scale-up to large units, together with the methods for transferring gradient separations from one scale to another are addressed. Noninvasive methods that have been developed for the physical characterization of various batches of SMC, which fulfill the regulatory requirements for cGMP production, are discussed. The applications of SMC for the separation and purification of large biomolecules, which demonstrate the full potential of this novel technology for an efficient downstream processing of biomolecules, are also presented.     

Electroosmotic pump‐supported molecularly imprinted monolithic column for capillary chromatographic separation of nitrophenol isomers

In this work, a novel molecularly imprinted polymer (MIP) monolithic column with integrated in‐column electroosmotic pump (EOP) was designed and successfully prepared to facilitate the capillary chromatography with MIP column. A silica‐based EOP was synthesized at the detection end of the MIP monolithic capillary column by so‐gel to provide the hydrodynamic driven force for the capillary chromatography. Because of large surface area and low fluidic resistance of the silica monolith,a strong and steady EOF was generated by silica‐based EOP, indicating that the EOP was quite compatible with MIP capillary column. With the sufficient EOF provided by EOP, the electro‐driven based capillary chromatographic separation of nitrophenol isomers was achieved in 4‐vinylpyridine‐based MIP monolithic capillary, which was originally proved infeasible because of the EOF shortage. No significant influence upon the specific recognition of the MIP was found due to the setting of EOP after the detection window of the column. The influence of experimental parameters on the EOF such as voltage and pH value of running buffer was investigated. The column was also evaluated by capillary liquid chromatographic mode to compare with EOP‐driven capillary chromatography. Higher column efficiency was obtained by EOP‐driven separation with improved peak shape. The results suggested that EOP‐supported technique would be a good way to solve the problem of weak EOF generation in electro‐driven capillary chromatography.     

Application of linear gradient elution in countercurrent chromatography for the separation of triterpenoid saponins from the roots of Pulsatilla koreana Nakai

A linear gradient elution method using countercurrent chromatography was developed for the separation of four triterpenoid saponins from the roots of Pulsatilla koreana Nakai, including hederacolchiside E, which is responsible for the neuroprotective activity of this plant. The target fraction was obtained by 80% methanol elution of solid phase column chromatography. The partition coefficients of the target compounds were very different, which means they are difficult to separate with a single biphasic solvent system. Several important parameters for gradient elution, such as addition of alcohol content to the solvent system, starting point of the second mobile phase, and the time for the gradient change were logically determined and optimized. Four triterpenoid saponins could ultimately be separated, analyzed by high‐performance liquid chromatography, and their structures were identified by comparing the mass spectra and NMR spectra with the literature data. The compounds and yields were: hederasaponin B ( 1 ; 21.3 mg/100 mg), hederacolchiside E ( 2 ; 19.8 mg/100 mg), cernuoside A ( 3 ; 18.4 mg/100 mg), and cernuoside B ( 4 ; 17.3 mg/100 mg). Gradient‐elution countercurrent chromatography allows the effective separation of compounds with a wide polarity range.     

梯度液相色谱中任意等度、线性和阶梯梯度组合下的保留时间公式

基于线性溶剂强度模型,应用特征线分析的方法求解梯度洗脱模式下的理想液相色谱模型。在考虑到梯度延迟时间会对溶质的保留时间造成影响的情况下,得到适合于梯度液相色谱中任意等度、线性和阶梯梯度组合条件下的保留时间推导公式。应用这些公式计算任意的梯度条件下的保留时间,并将得到的结果与数值计f算的结果进行比较,二者完全一致,从而验证了推导得到的保留时间公式的正确性。由于这些公式具有形式简单、适用范围广等优点,因此可方便地应用于实际应用中,具有较高的实用价值。     

反相高效液相色谱双梯度洗脱分离肽混合物及质谱分析

复杂肽段混合物的有效分离是高覆盖率地鉴定蛋白质混合物的前提。“鸟枪法”(Shotgun)蛋白质组学研究策略通常采用蛋白酶切、二维液相色谱-串联质谱分析肽段混合物从而鉴定蛋白质,其中高效率地分离肽段混合物是关键步骤之一。本文通过pH梯度结合有机溶剂梯度的反相高效液相色谱(RP-HPLC)进行一维液相色谱分离,按等时间间隔收集馏分并将一个梯度的前段的一个馏分与后段一个馏分混合,然后进行纳升级液相色谱-质谱联用(nanoRPLC-MS/MS)分析。将该方法应用于酵母蛋白质的分离和鉴定,实验结果为: 与常规的强阳离子色谱-反相液相色谱-质谱分离鉴定方法相比,采用pH梯度结合有机相梯度的RP-HPLC-RPLC-MS分离鉴定方法多鉴定到567个酵母蛋白质(簇,含有3035个唯一肽段);其中鉴定到肽段的pI分布范围为3.42～12.01,相对分子质量范围为587.67～3499.79;蛋白质的pI分布范围为3.82～12.19,相对分子质量范围为3446.55～432905。该结果表明这种方法在复杂体系蛋白质组分离鉴定中具有明显的优势,在蛋白质组学研究中有较好的应用前景。     

Polymeric cation-exchange monolithic columns containing phosphoric acid functional groups for capillary liquid chromatography of peptides and proteins

Two different monoliths, both containing phosphoric acid functional groups and polyethylene glycol (PEG) functionalities were synthesized for cation-exchange chromatography of peptides and proteins. Phosphoric acid 2-hydroxyethyl methacrylate (PAHEMA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) were reacted with polyethylene glycol diacrylate (PEGDA) and polyethylene glycol acrylate (PEGA), respectively, in 75-μm i.d. UV-transparent fused-silica capillaries by photo-initiated polymerization. The hydrophobicities of the monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. The resulting monoliths exhibited lower hydrophobicities than strong cation-exchange monoliths previously reported using PEGDA as cross-linker. Dynamic binding capacities of 31.2 and 269 mg/mL were measured for the PAHEMA–PEGDA and BMEP–PEGA monoliths, respectively. Synthetic peptides were eluted from both monoliths in 15 min without addition of acetonitrile to the mobile phase. Peak capacities of 50 and 31 were measured for peptides and proteins, respectively, using a PAHEMA–PEGDA monolith. The BMEP–PEGA monolith showed negligible hydrophobicity. A peak capacity of 31 was measured for the BMEP–PEGA monolith when a 20-min salt gradient rate was used to separate proteins. The effects of functional group concentration, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64) reproducibilities were achieved. The performance of the monoliths in ion-exchange separation of peptides and proteins was superior to other polymeric monolithic columns reported previously when organic solvents were not added to the mobile phase.     

梯度液相色谱保留时间公式在梯形梯度洗脱研究中的应用

根据前期得到的梯度液相色谱保留时间计算公式,在不指定溶剂强度模型形式的前提下,探讨了梯形梯度洗脱的一些特点。对于溶质在梯形梯度坡度上流出时的情形,推导得到溶质流出色谱柱所对应的流动相组成(φ_R)随梯度斜率(B)变化的表达式。该公式表明,在该情形中φ_R将会随着B值的增加而增加。对于溶质在梯形梯度最后一个等度区间流出时的情形,如果初始和终止流动相组成保持不变而仅有梯度的斜率发生变化时,从理论上证明了溶质保留时间(t_R)与梯度斜率的倒数(1/B)之间呈线性关系。实验中以C18色谱柱为固定相,甲醇-水为流动相,联苯为样品,测定了不同流动相组成以及梯形梯度条件下的保留时间,所得到的实验值与理论值吻合,从而验证了理论方法的正确性。     

Theoretical background of multicomponent gas chromatography for high concentrations

Summary The paper presents a classification of various theoretical models for multicomponent gas chromatography at u=var for high concentrations of the mixture components with consideration of the major distinctive features of the interphase mass exchange inside the porous grains of the adsorbent and outside them within the porous medium of the chromatographic column. It has been shown that the conditions determining a given front behaviour of multicomponent gas mixture at u=var depend on: theoretical models for the kinetics and dynamics of adsorption; the values of mass exchange parameters inside the porous grains and in the porous medium. Formulae have been derived for calculating the values of the quantities characterising the frontal behaviour in the stage of adsorption and desorption of multicomponent mixture at u=var.     

Dynamic ion-exchange chromatography with post-column reaction detection for the determination of the rare earth elements in monazites

Summary Separation and determination of lanthanum, cerium, praseodymium, neodymium and samarium in monazites have been achieved by dynamic ion-exchange chromatography. The ore samples are decomposed by sulfuric acid and the rare earths are separated in a group as oxalates. The rare earth elements are then separated from each other on a column of bonded phase silica by gradient elution with 0.05 to 0.5 M lactic acid (pH 3.5) in the presence of 0.01 M sodium 1-octanesulfonate. Post-column reaction with Arsenazo III is used for detection and quantification of the individual rare earth elements. Results are quoted for lanthanum, cerium, praseodymium, neodymium and samarium in monazites. Detection limit is 1 μg ml⁻¹ with a S/N ratio of 3. The separation is complete within 27 min valley to valley resolution. Precision of better than 1% can usually be obtained.