Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. II. Separation of amino acids and peptides on sulfonated naphthalimido-modified silyl silica gel

The potential of 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP) as a mixed-mode stationary phase for capillary electrochromatography (CEC) was investigated for the separation of charged analytes, taking four amino acids (tyrosine, phenylalanine, tryptophan, histidine) as model analytes. The elution process of these charged analytes in CEC with SNAIP was dominated by a combination of both electrophoretic process and chromatographic process involving hydrophobic as well as electrostatic interactions. In order to study the retention mechanism, the CEC retention factor k* and the velocity factor ke* were measured for the amino acids, which allowed the assessment of the respective contribution from the differential processes underlying the separation. Migration and retention could be mediated by changing various mobile phase compositions, including buffer pH, buffer concentration, and concentration of organic solvent. Based on the results obtained by separation of the amino acids, the separation of eight peptides (Gly-Val, Gly-Phe, Gly-Ile, Gly-His, Gly-Lys, Lys-Lys, Gly-Gly-Gly, Gly-Gly-His) was attempted. A good separation was achieved under an isocratic elution with a mobile phase consisting of 35 mM phosphate buffer (pH 3.8) and 40% methanol.     

Investigation of a novel mixed-mode stationary phase for capillary electrochromatography. Part III: Separation of nucleosides and nucleic acid bases on sulfonated naphthalimido-modified silyl silica gel

Capillary electrochromatography (CEC) with a novel stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), proved useful for the separation of nucleosides and nucleic acid bases. The application scope of SNAIP, which is a relatively polar reversed-phase (RP)-type stationary phase, was successfully expanded to include the CEC separation of polar compounds although the combination of non-polar RP phase with highly aqueous mobile phase is often inadequate. Due to the permanently charged sulfonic acid groups and the naphthalimidopropyl moiety, the retention of charged and relatively polar nucleosides as well as bases on the SNAIP stationary phase was effected by electrostatic and hydrophobic interactions. This yielded a unique selectivity on SNAIP toward nucleosides and bases. The characteristic EOF on SNAIP, which was stronger at higher aqueous content in the mobile phase, proved suitable for the separation of polar compounds in reversed-phase mode with highly aqueous mobile phase. In addition, when a double stepwise gradient was employed to accelerate the latest peak (adenine), the elution time was shortened to less than half its original duration.     

离线pH梯度-强阳离子交换色谱法分离肽段混合物

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。Shotgun蛋白质组学研究通常采用二维液相色谱(强阳离子交换色谱-反相色谱)分离后接串联质谱检测的方法。但由于离子交换色谱体系中含有高浓度的盐,使得在线分析的难度较大;而在离线分析时,也常因需要对高盐组分进行脱盐处理而易引起样品损失。因此,该文尝试用pH梯度替代盐梯度,实现pH梯度-强阳离子交换色谱方法应用于复杂肽段混合物的分离。通过对缓冲体系pH值的计算,优化了乙酸-乙酸铵体系线性pH梯度配合盐梯度的离子交换色谱方法,以及柠檬酸-氨水体系线性pH梯度的离子交换色谱方法。将这两种方法应用于牛血清白蛋白酶切产物的分离取得了与常规强阳离子交换色谱相似的分离效果。乙酸-乙酸铵体系采用的是低浓度的可挥发性铵盐,采用真空冻干的方法可以有效除盐,基质辅助激光解吸质谱靶上自然挥发也可以达到较好的脱盐效果,简化了常规方法繁琐费时的脱盐步骤及避免了由此造成的样品损失。柠檬酸-氨水体系采用pH梯度洗脱替代盐梯度洗脱,大大降低了体系中的盐浓度。这两种方法在复杂体系蛋白质组研究的样本预分离中具有较好的应用前景。     

Investigation of the novel mixed-mode stationary phase for capillary electrochromatography. I. Preparation and characterization of sulfonated naphthalimido-modified silyl silica gel

A novel packing material, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP), was prepared for the use as a stationary phase of capillary electrochromatography (CEC). The sulfonic acid groups on SNAIP stationary phase contributed to the generation of electroosmotic flow (EOF) at low pH and served as a strong cation-exchanger. In CEC with SNAIP, a mixed-mode separation was predicted, comprising hydrophobic and electrostatic interactions as well as electrophoretic migration process. In order to understand the retention mechanism on SNAIP, effects of buffer pH, concentration, and mobile phase composition on EOF mobility and the retention factors of barbiturates and benzodiazepines were systematically investigated. Moreover, the retention behavior of barbiturates on SNAIP was investigated and compared with those on octadecyl silica (ODS), phenyl-bonded silica, and 3-(1,8-naphthalimido)propyl-modified silyl silica gel to confirm the presence of pi-pi interaction on its retention mechanism. It was observed that a column efficiency was more than 85,000 N/m for retained compounds and the relative standard deviations for the retention times of EOF marker, thiourea, and five barbiturates were below 2.5% (n = 4). Under an applied voltage of 20 kV and a mobile phase consisted of 5 mM phosphate (pH 3.8) and 40% methanol, the baseline separation of five barbiturates was achieved within 3 min.     

Characterization of unstable ion-exchange chromatographic separation of proteins.

Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (beta-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within +/-0.1 unit and the ionic strength within +/-0.002 mol/l in order to meet the criteria (+/-5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained.     

全二维微柱液相色谱-质谱鉴定人胃癌组织中的差异蛋白质

构建了以阳离子交换色谱-反相色谱(SCX-RPLC)为分离模式的新型全二维微柱液相色谱-质谱分离平台.采用了醋酸铵缓冲液梯度洗脱,实现了第一维肽段的分步洗脱,洗脱的肽段经富集除盐后通过接口进入反相色谱微柱,通过线性梯度实现第二维进一步分离,最后进入质谱进行检测.采用此平台分析了人胃癌组织与正常组织提取蛋白质信息,其中正常胃组织鉴定蛋白质数为537个,而癌症组织鉴定蛋白质数目为506个.对胃癌和正常组织两种提取蛋白质酶解产物的蛋白质检索结果进行比较分析,将鉴定的蛋白质按照物理性质进行分布,找出正常组织与癌症组织间蛋白质差异,筛选出一种可能发生变异的癌症特有蛋白.     

Fractionation and separation of human salivary proteins by pH-gradient ion exchange and reversed phase chromatography coupled to mass spectrometry

In the present work, a 2-D capillary liquid chromatography method for fractionation and separation of human salivary proteins is demonstrated. Fractionation of proteins according to their pI values was performed in the 1-D employing a strong anion exchange (SAX) column subjected to a wide-range descending pH gradient. Polystyrene-divinylbenzene (PS-DVB) RP columns were used for focusing and subsequent separation of the proteins in the 2-D. The SAX column was presaturated with a high pH buffer (A) consisting of 10 mM amine buffering species, pH 9.0, and elution was performed with a low pH elution buffer (B) having the same buffer composition and concentration as buffer A, but pH 3.5. Isoelectric point fractions eluting from the 1-D column were trapped on PS-DVB trap columns prior to back-flushed elution onto the PS-DVB analytical column for separation of the proteins. The 1-D fraction eluting at pH 9.0-8.7 was chosen for further analysis. After separation on the RP analytical column, nine RP protein fractions were collected and tryptic digested for subsequent analyses by MALDI TOF MS and column switching capillary LC coupled to ESI TOF MS and ESI QTOF MS. Eight proteins and two peptides were identified in the pH 9.0-8.7 fraction using peptide mass fingerprinting and uninterpreted MS/MS data.     

Optimization of monoclonal antibody purification by ion-exchange chromatography. Application of simple methods with linear gradient elution experimental data

Simple methods for the optimization of ion-exchange chromatography of proteins in our previous papers were applied to cation-exchange chromatography purification of monoclonal antibodies (Mab). We carried out linear gradient elution experiments, and obtained the data for the peak salt concentration and the peak width. From these data, the distribution coefficient as a function of salt concentration, and the height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity were calculated. The optimized linear gradient elution conditions were determined based on the relationship between buffer consumption and separation time. The optimal stepwise elution conditions were determined based on the relationship between the distribution coefficient and the salt concentration.     

Effect of buffer concentration on gradient chromatofocusing performance separating protiens on a high-performance DEAE column

Gradient chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta-lactoglobulin A and B in gradient chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient chromatofocusing technique over the conventional chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, beta-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of beta-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.     

Stepwise gradient of linear polymer matrices in microchip electrophoresis for high-resolution separation of DNA

The stepwise gradient of linear polymer matrices in microchannel electrophoresis is proposed as a means of achieving high-resolution separation of DNA samples containing a wide range of fragment sizes. In this method, multiple discrete steps in terms of polymer type or concentration are created in the microchannel by injecting appropriate solutions in order. The mixing of the various steps is found to be negligible compared to the effective length of separation channel, confirming that a stepwise gradient of matrices is formed. This technique is successfully applied to the analysis of restriction digest fragments and DNA ladders, and is demonstrated to provide higher resolution than the isocratic method, for both small and large fragments simultaneously. Even though the stepwise gradient is created manually, the reproducibility of the migration times of fragments in DNA samples is found to be quite good. Taken the separation of 100 bp DNA ladder in three steps gradient pattern as an example, the relative standard deviations of migration times are respectively less than 0.53% and 3.1% in six consecutive injections in one channel and in different channels. The migration of DNA fragments in gradient mode is shown to be similar to that for the isocratic scheme, allowing the design of each step to be made in reference to existing knowledge. These promising results indicate the great potential of this stepwise gradient method for the analysis of DNA by microchip electrophoresis, offering both high resolution and good reproducibility.     

Analysis of synthetic peptides by capillary electrophoresis: effect of organic solvent modifiers and variable electrical potentials on separation efficiencies

In this study, procedures based on volatile ammonium acetate buffer electrolytes of high pH value containing different organic solvent modifiers have been developed to achieve very high efficiency separations of histidine-containing synthetic peptides by high-performance capillary electrophoresis (HPCE) employing untreated fused silica capillaries. Different organic solvents, including acetonitrile, methanol and ethanol, at high volume fractions were used to modify the composition of the background buffer electrolyte. With the peptides investigated, it was found that methanol had the greatest effect in terms of enhancement of separation efficiency, as determined from the evaluation of theoretical plate numbers, N, of these HPCE systems. On the other hand, separation selectivities, e.g. the alpha(ij) values, did not change significantly as the volume fraction, psi, of the organic solvents was increased up to psi = 60% (v/v). Under these conditions, very rapid, e.g. 1-2 min, separation times could be still achieved. Compared to the effect of carrying out the separation of these peptides at constant voltage, a dramatic increase in the separation efficiency was also achieved by applying a linear voltage gradient during the HPCE experiment. Under optimal conditions of organic solvent composition and linear voltage gradient ramps, very high peak efficiencies for the studied set of synthetic peptides with N values of approximately 2-3 million theoretical plates per meter could be routinely obtained with fast analysis times. Moreover, these buffer electrolyte conditions are compatible with direct interfacing of the HPCE effluent to electrospray ionisation and ion trap mass spectrometers, thus expanding the analytical capabilities of these HPCE systems.     

Separation of peptides by strong cation-exchange capillary electrochromatography

Separation of small peptides on ion-exchange capillary electrochromatography (IE-CEC) with strong cation-exchange packing (SCX) as stationary phase was investigated. It was observed that the number of theoretical plates for small peptides varied from 240000 to 460000/m, and the relative standard deviation for t0 and the migration time of peptides were less than 0.57% and 0.27%, respectively for ten consecutive runs. Unusually high column efficiency has been explained by the capillary electrophoretic stacking and chromatofocusing phenomena during the injection and separation of positively charged peptides. The sample buffer concentration had a marked effect on the column efficiency and peak area of the retained peptides. The influences of the buffer concentration and pH value as well as the applied voltage on the separation were investigated. It has been shown that the electrostatic interaction between the positively charged peptides and the SCX stationary phase played a very important role in IE-CEC, which provided the different separation selectivity from those in the capillary electrophoresis and reversed-phase liquid chromatography. A fast separation of ten peptides in less than 3.5 min on IE-CEC by adoption of the highly applied voltage was demonstrated.     

Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

The design and synthesis of alanine-based indolicidin derivatives with identical physicochemical properties and their separation using capillary electrophoresis

Four novel alanine-based indolicidin peptide derivatives were designed containing one WPW motif and two alanine residues, resulting in peptides of similar sequence. The separation of these peptides with identical physicochemical properties including molar mass, charge, and secondary structure as characterized by circular dichroism spectroscopy is very difficult; and the separation of peptides with differing physicochemical properties has only previously been reported. Capillary electrophoresis parameters such as separation buffer concentration, separation buffer pH, capillary length, and separation voltage were investigated to optimize the analysis. Using optimized conditions of a background electrolyte containing 5 mM formic acid of pH 2.0, total capillary length of 51 cm and a voltage of 10 kV enabled a baseline separation of the four peptides. The relative standard deviation of the peak areas and migration times for method repeatability (n = 3) were found to be lower than 8% and 3%, respectively. In addition, reasoning for the separation of these peptides is proposed based on the acidity of the formic acid buffer and the hydrophobic grouping of the tryptophan residues in the peptide primary sequence.     

反相高效液相三元流动相台阶梯度分离条件的快速优化方法

 根据溶质在柱内的迁移规律 ,建立了一种利用线性梯度实验快速获得溶质保留值方程系数 ,然后以串行响应函数为优化指标进行多台阶梯度分离条件优化的方法。与利用等度实验获得保留值方程的方法相比 ,该法可以大大缩短优化时间。通过该方法对芳香胺和衍生化氨基酸样品进行了分离 ,获得了满意的分离度 ,表明该方法的预测精度很好。     

Capillary electrophoretic and micellar electrokinetic separations of asymmetric dimethyl-L-arginine and structurally related amino acids: quantitation in human plasma

We report the development of efficient electrophoretic methods for the separation and quantification of L-arginine and six naturally occurring derivatives that are structurally and functionally related. Capillary electrophoresis (CE) employing a concentrated borate buffer at pH 9.4 achieves the separation of mixtures containing dimethyl-L-arginine, NG-monomethyl-L-arginine, L-arginine, L-homoarginine, L-ornithine, and L-citrulline as 4-fluoro-7-nitrobenzofurazan derivatives. In addition, the separation of the isomeric dimethyl-L-arginine derivatives (symmetric and asymmetric) is attained with baseline resolution by micellar electrokinetic chromatography (MEKC) when a high concentration of deoxycholic acid is added as a surfactant to the same running buffer. The influence of buffer type, concentration, and pH on the separation was studied to optimize separation conditions. The limit of quantitation (LOQ) for asymmetric dimethyl-L-arginine in aqueous solution was determined to be 20 microM using UV absorption in a CE separation and 0.1 microM using laser induced fluorescence (LIF) detection in an MEKC separation. This newly developed method was successfully applied for the quantitation of asymmetric dimethyl-L-arginine and L-arginine in human plasma samples at levels that might be used as a clinical diagnostic for cardiovascular disease (0.125 microM LOQ).     

黄芪中黄酮类组分的高效液相色谱分离条件的快速优化

以黄芪为研究对象,对黄芪中乙酸乙酯部位的化学成分进行了高效液相色谱分析并对其色谱操作条件进行了快速优化。根据几个线性梯度下的保留时间来计算各组分的保留参数,然后利用重叠分辨图法确定其最佳分析条件。在选定的最佳条件下各组分分离情况良好。利用梯度保留时间计算保留参数比较方便快速,并可以有效地避免以往等度线性回归法遇到的峰识别问题。该方法更适用于实际复杂样品色谱分析条件的优化。     

Separation of Peptides by Pressurized Capillary Electrochromatography

A novel gradient pressurized capillary electrochromatography (pCEC) instrument was developed to separate peptides.Two gradient elution modes,hydrophobic and hydrophilic interaction mode in pCEC,were performed on this instrument.Baseline separation of six peptides was obtained on two gradient modes with C18 column and strong cationic exchange column respectively.The effects of mixer volume and total frow rate of pumps on resolution were also discussed.     

A macrocyclic polyamine as an anion receptor in the capillary electrochromatographic separation of carbohydrates

An analytical approach of the 32-membered macrocyclic polyamine, 1,5,9,13,17,21,25,29-octaazacyclodotriacontane ([32]ane-N8) was described for the capillary electrochromatographic (CEC) separation of derivatized mono- and disaccharides. The column displayed reversal electroosmotic flow (EOF) at pH below 7.0, while a cathodic EOF was shown at pH above 7.0. The reductive amination of saccharides was carried out with p-aminobenzoic acid. Some parameters that affect the CEC separations were investigated. Several competitive ligands, such as Tris, EDTA and phosphate were also examined for the effect on the performance. We achieved a complete separation of all compounds as well as the excess derivatizing agent by using borate buffer (pH 9.0) in a mode of concentration gradient (60 mM inlet side and 70 mM outlet side). The relative standard deviation of the retention time measured for each sample was less than 4% in six continuous runs, suggesting that the bonded phase along with the gradient formed inside the column was quite stable. With the mixing modes of anion coordination, anion exchange, and shape discrimination, the interaction adequately accomplishes the separation of carbohydrates which are epimers or have different glycosidic linkage, although the electrophoretic migration is also involved in the separation mechanism.     

Separation of structurally related synthetic peptides by capillary zone electrophoresis

The separation of two different sets of synthetic peptides has been investigated by high-performance capillary zone electrophoresis utilising naked, fused silica capillaries. The effects of electrolyte pH, buffer concentration, capillary length and electric field strength on the separation efficiency and selectivity were systematically varied, with the highest resolution achieved with buffer electrolytes of low pH and relatively high ionic strength. Under optimised separation conditions utilising the "short end injection" separation approach with negative electric field polarity, a series of eight structurally-related synthetic peptides were baseline resolved within 4 min without addition of any modifier of the background electrolyte with separation efficiencies in the vicinity of 600000 theoretical plates/m. Further significant enhancement of separation efficiencies could be achieved by taking advantage of the "long end injection" approach with positive electric field polarity. The outcome of these experimental variations parallels the "sweeping" effect that has been observed in the capillary electrochromatographic and micellar electrokinetic separations of polar molecules and permits rapid resolution of peptides with focusing effects. In addition, small changes in the electrolyte buffer pH and concentration were found to have a significant impact on the selectivity of synthetic peptides of similar intrinsic charge. These observations indicate that multi-modal separation mechanisms operated under these conditions with the unmodified fused silica capillaries. This study, moreover, documents additional examples of peptide-specific multi-zoning behaviour in the high-performance capillary zone electrophoretic separation of synthetic peptides.