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.     

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.     

On-chip sample pretreatment using a porous polymer monolithic column for solid-phase microextraction and chemiluminescence determination of catechins in green tea

A porous polymer monolithic column for solid-phase microextraction and chemiluminescence detection was integrated into a simple microfluidic chip for the extraction and determination of catechins in green tea. The porous polymer was prepared by poly(glycidyl methacrylate-co-ethylene dimethacrylate) and modified with ethylenediamine. Catechins can be concentrated in the porous polymer monolithic column and react with potassium permanganate to give chemiluminescence. The microfluidic chip is reusable with high sensitivity and very low reagent consumption. The on-line preconcentration and detection can be realized without an elution step. The enrichment factor was calculated to be about 20 for catechins. The relative chemiluminescence intensity increased linearly with concentration of catechin from 5.0 × 10(-9) to 1.0 × 10(-6) M and the limit of detection was 1.0 × 10(-9) M. The proposed method was applied to determine catechin in green tea. The recoveries are from 90% to 110% which benefits the actual application for green tea samples.     

Rapid determination of molecular parameters of synthetic polymers by precipitation/redissolution high‐performance liquid chromatography using “molded” monolithic column

Rapid high‐performance liquid chromatography (HPLC) of polystyrenes, poly(methyl methacrylates), poly(vinyl acetates), and polybutadienes using a monolithic 50 × 4.6 mm i.d. poly(styrene‐co‐divinylbenzene) column have been carried out. The separation process involves precipitation of the macromolecules on the macroporous monolithic column followed by progressive elution utilizing a gradient of the mobile phase. Depending on the character of the separated polymer, solvent gradients were composed of a poor solvent such as water, methanol, or hexane and increasing amounts of a good solvent such as THF or dichloromethane. Monolithic columns are ideally suited for this technique because convection through the large pores of the monolith enhances the mass transport of large polymer molecules and accelerates the separation process. Separation conditions including the selection of a specific pair of solvent and precipitant, flow rate, and gradient steepness were optimized for the rapid HPLC separations of various polymers that differed broadly in their molecular weights. Excellent separations were obtained demonstrating that the precipitation‐redissolution technique is a suitable alternative to size‐exclusion chromatography (SEC). The molecular weight parameters calculated from the HPLC data match well those obtained by SEC. However, compared to SEC, the determination of molecular parameters using gradient elution could be achieved at comparable flow rates in a much shorter period of time, typically in about 1 min. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2767–2778, 2000     

Purification of the glycoprotein glucose oxidase from Penicillium amagasakiense by high-performance liquid chromatography

Fast protein liquid chromatography (FPLC) in combination with ion-exchange chromatography on a Mono Q column was used to purify glucose oxidase from Penicillium amagasakiense to homogeneity. Purification was performed with a mixed pH and salt gradient, with 20 mM phosphate buffer (pH 8.5) as starting buffer (A) and 50 mM acetate buffer (pH 3.6) with 0.1 M NaCl as elution buffer (B). Elution conditions were optimized to permit the simultaneous purification and separation of the glucose oxidase isoforms. Three peaks, each consisting of 1-2 isoforms and exhibiting a homogeneous titration curve profile, were resolved with a very flat linear gradient of 5.0-5.1% B in 40 ml. Three more peaks, each consisting of several isoforms, were eluted at 10%, 30% and 100% B. Optimization of the elution conditions and separation of the glucose oxidase isoforms was only possible because of the rapidity of each purification step and the high resolution provided by FPLC and Mono Q.     

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.     

Highly efficient monolithic silica capillary columns modified with poly(acrylic acid) for hydrophilic interaction chromatography

Monolithic silica capillary columns for hydrophilic interaction liquid chromatography (HILIC) were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained the high permeability (K=5 x 10(-14)m(2)) and provided a plate height (H) of less than 10 microm at optimum linear velocity (u) and H below 20 microm at u=6mm/s for polar solutes including nucleosides and carbohydrates. The HILIC mode monolithic silica capillary column was able to produce 10000 theoretical plates (N) with column dead time (t(0)) of 20s at a pressure drop of 20 MPa or lower. The total performance was much higher than conventional particle-packed HILIC columns currently available. The gradient separations of peptides by a capillary LC-electrospray mass spectrometry system resulted in very different retention selectivity between reversed-phase mode separations and the HILIC mode separations with a peak capacity of ca. 100 in a 10 min gradient time in either mode. The high performance observed with the monolithic silica capillary column modified with poly(acrylic acid) suggests that the HILIC mode can be an alternative to the reversed-phase mode for a wide range of compounds, especially for those of high polarity in isocratic as well as gradient elution.     

Analyses of sulfonamide antibiotics in meat samples by on-line concentration capillary electrochromatography-mass spectrometry

In this work, a series of poly(divinylbenzene-alkyl methacrylate) monolithic stationary phases, which were prepared by single step in situ polymerization of divinylbenzene and various alkyl methacrylates (butyl-, octyl-, lauryl- or stearyl methacrylate), were developed as separation columns of nine common sulfonamide antibiotics for capillary electrochromatography (CEC) coupled to mass spectrometry (MS). Results indicated that the sulfonamide's retention became weak with increased carbon chain length of alkyl methacrylate monomer (for example, t(R)=68 min and 21 min for butyl- and lauryl methacrylate, respectively). Among them, the poly(divinylbenzene-octyl methacrylate) (poly(DVB-OMA)) monolith was regarded as the optimal separation column as this provided better resolution within the shortest retention time. Moreover, the cross-sectional roughness of the monolithic column-end, that was used to couple to the ESI interface, strongly influenced the electrospray stability in the CEC-MS. Before the column was connected to the ESI-MS, a simple polishing was done to reduce the roughness of the column end that resulted to a great improvement in the signal stability. The relative standard deviations (RSDs) of the peak areas for the unpolished and polished ends of the poly(DVB-OMA) columns (n=5) were in the range of 46.1-60.2% and 8.9-16.4%, respectively. Furthermore, optimization of the mobile phase composition and the gradient elution strategy successfully determined the sulfonamide antibiotics in meat samples with as low as 10 μg/L level.     

Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection

Water- and fat-soluble vitamins were separated on a MetaChem Polaris C18-A (150 mm×4.6 mm, 3 μm particle size) in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of 0.010% trifluoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the flow rate 0.7 ml min⁻¹. A linear gradient profile (A:B) started at 95:5 and was constant in the first 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and finally linearly increased up to 95:5 ratio of water phase in the last 5 min of the separation. The most suitable detection wavelength for simultaneous vitamin determination was 280 nm. The method was applied for the solid sample of pharmaceutical preparation (B-Komplex), fortified powdered drinks (multi-vitamin) and food samples. The results were in good agreement with the declared values.     

利用HCI程序确定儿茶酚化合物的反相高效液相色谱最佳分离条件

有效地确定了反相高效液相色谱分离儿茶酚化合物的最佳条件。在水和甲醇的二元流动相里分别加入乙酸缓冲液,利用基于ln k=ln kw +SF, k=A+B/F, ln k=L+MF+NF2 (F是流动相中有机物甲醇的体积分数)等保留因子的一次或二次方程式的塔板理论得到色谱分离结果;利用保留原理得到等度和梯度洗脱的最佳条件。得出最佳初始流动相是含0.1%乙酸的水和含0.1%乙酸的甲醇(体积比为75∶25)的混合溶液;梯度洗脱条件：初始流动相保持15 min,然后用10 min的时间将上述二元流动相的体积比线性变换成50∶50,直到完成全部分离。通过实验证实该计算结果与实验值相近。     

Analysis of flavonoids in leaves of Adinandra nitida by capillary electrochromatography on monolithic columns with stepwise gradient elution

Extracts of Adinandra nitida leaves, known as Shiyacha in China, were analyzed by monolithic columns of capillary electrochromatography (CEC). To obtain good resolution within a short time, stepwise gradient elution of CEC was employed, and the effects of experimental parameters, such as the buffer, the gradient conditions, and the mode of injection were studied systematically. Under optimized conditions, analysis could be accomplished in 25 min on a monolithic rod of macroporous poly(butyl methacrylate-co-ethylene dimethacrylate). With two identified flavonoids, epicatechin and apigenin, as markers, a quality control method for Shiyacha and its relevant products was established. The calibration curves exhibited good linear behavior over the concentration range of two orders of magnitude. On combination with an on-line concentration technique, the detection limit of flavonoids could be decreased to 25 ng for apigenin.     

Preparation and evaluation of poly(alkyl methacrylate-co-methacrylic acid-co-ethylene dimethacrylate) monolithic columns for separating polar small molecules by capillary liquid chromatography

In this study, methacrylic acid (MAA) was incorporated with alkyl methacrylates to increase the hydrophilicity of the synthesized ethylene dimethacrylate-based (EDMA-based) monoliths for separating polar small molecules by capillary LC analysis. Different alkyl methacrylate–MAA ratios were investigated to prepare a series of 30% alkyl methacrylate–MAA–EDMA monoliths in fused-silica capillaries (250-μm i.d.). The porosity, permeability, and column efficiency of the synthesized MAA-incorporated monolithic columns were characterized. A mixture of phenol derivatives is employed to evaluate the applicability of using the prepared monolithic columns for separating small molecules. Fast separation of six phenol derivatives was achieved in 5 min with gradient elution using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. In addition, the effect of acetonitrile content in mobile phase on retention factor and plate height as well as the plate height-flow velocity curves were also investigated to further examine the performance of the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column. Moreover, the applicability of prepared polymer-based monolithic column for potential food safety applications was also demonstrated by analyzing five aflatoxins and three phenicol antibiotics using the selected poly(lauryl methacrylate-co-MAA-co-EDMA) monolithic column.     

Separation of proteins by hydrophobic interaction chromatography at low salt concentration

We investigated protein separation by hydrophobic interaction chromatography (HIC) at low salt concentration on the supports of various hydrophobicities. Hydrophobic proteins could be successfully separated with more than 90% recovery by gradient elution of ammonium sulfate from 0.3-0.5 M to 0 in 50 mM phosphate buffer (pH 6.8) by using supports whose hydrophobicities were properly adjusted individually for each protein. Satisfactory results were also obtained by isocratic elution without ammonium sulfate and gradient elution of ethanol from 0 to 10%. HIC at low salt concentration was compatible with other modes of liquid chromatography like ion-exchange chromatography. On the other hand, it was not successful to separate hydrophilic proteins at low salt concentration. Recoveries of hydrophilic proteins decreased before they were retained enough as support hydrophobicity increased. Therefore, it is inevitable to use a higher concentration of salt, e.g., 1-2 M ammonium sulfate, on hydrophilic or moderately hydrophobic support in order to retain hydrophilic proteins without decrease in recovery.     

Modeling of salt and pH gradient elution in ion‐exchange chromatography

The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion‐exchange columns is a well‐established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO₃ (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well‐characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.     

Overloaded gradient elution chromatography on heterogeneous adsorbents in reversed-phase liquid chromatography

Overloaded band profiles of phenol were measured on a C18-Kromasil column in gradient elution conditions. The mobile phase used was a mixture of methanol and water. The volume fraction of methanol was allowed to vary between 0 and 0.5. A general adsorption model, which expresses the amount of phenol adsorbed q* as a function of both its concentration C and the composition phi of the organic modifier (methanol) in the mobile phase, was empirically derived from previous independent adsorption experiments based on frontal analysis (FA) and frontal analysis by the characteristic point (FACP). Accordingly, the general model was an extension of the simplest heterogeneous model, the Bilangmuir model, to non-isocratic conditions. The low-energy sites followed the classical linear solvent strength model (LSSM), but not the high-energy sites whose saturation capacity linearly decreased with phi. The general model was validated by comparing the experimental and simulated band profiles in gradient elution conditions, in linear and non-linear conditions, as well. The band profiles were calculated by means of the equilibrium-dispersive model of chromatography with a finite difference algorithm. A very good agreement was observed using steps gradient (delta phi) from 0 to 50% methanol and gradient times t(g) of 20, 25, 30, 40, 60, 80 and 100 min. The agreement was still excellent for steps gradient from 5 to 45% (t(g) = 25 min), 5 to 35% (t(g) = 50 min), 5 to 25% (t(g) = 50 min) and 5 to 15% (t(g) = 50 min). Significative differences appeared between experience and simulation when the slope of the gradient (delta phi/t(g)) became too strong beyond 3.3% methanol per minute. This threshold value probably mirrored the kinetic of arrangement of the G18-bonded chains when the methanol content increased in the mobile phase. It suggested that the chromatographic system was not in a full thermodynamic equilibrium state when very steep mobile phase gradients were applied.     

Development of a perfusion ion-exchange chromatography method for the separation of soybean proteins and its application to cultivar characterization

A perfusion ion-exchange chromatography method has been designed, for the first time, for the separation of soybean proteins and its application to the characterization of soybean cultivars. For that purpose, the gradient, the mobile phase composition (buffer concentration, buffer pH, and elution salt), and the temperature were optimized. The method consisted of a two-step gradient (0% B for 2 min and from 0 to 50% B in 10 min) being mobile phase A a 2 0mM borate buffer (pH 9) and mobile phase B a 20 mM borate buffer (pH 9) containing 1M sodium chloride. The procedure used for the preparation of sample solutions was significantly simpler than that proposed by other authors and basically consisted of dissolving in water. This method enabled the separation of soybean proteins from a soybean protein isolate in 11 peaks in about 9 min. The method was used to separate soybean proteins in different commercial soybeans. In general, the 11 peaks yielded by the soybean protein isolate were also observed in the chromatograms of all soybeans. However, the area percentages of every peak in every soybean enabled the differentiation between soybeans. Moreover, the method was also used to separate soybean proteins in the proteic fractions obtained from every soybean. Multivariate methods were used for patterns recognition and the classification of samples.     

阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

3-氨基-9-乙基咔唑衍生化寡糖混合物的高效液相色谱分离及激光解吸电离飞行时间质谱分析

建立了以3-氨基-9-乙基咔唑(AEC)为衍生化试剂对寡糖的标记方法。寡糖的还原端与AEC的伯氨基反应生成烯胺,再被NaBH3CN还原为二级胺,使得寡糖被AEC标记。衍生物通过反相高效液相色谱分离纯化,采用的色谱柱为Waters Symmetry C18柱(3.9 mm×150 mm,5 μm),乙腈和乙酸铵水溶液(pH 4.5)为流动相,梯度洗脱,在254 nm波长处检测,并以基质辅助激光解吸电离飞行时间质谱进行分析。在此衍生化条件和色谱条件下,葡寡糖衍生物分离良好,并且AEC衍生可显著提高葡寡糖的质谱检测灵敏度。该方法适用于寡糖的分离纯化和结构分析,并与生物质谱具有良好的兼容性,表明该方法在微量寡糖链分析方面有广阔的应用前景。