Residence time distribution study for the catalytic packing MULTIPAK®

Residence time distribution (RTD) experiments were carried out using the catalytic packing MULTIPAK® in a 250 mm inner diameter column. The axial dispersion coefficients and dynamic liquid hold-up were derived from the RTD curves. Both hold-up and axial Péclet number were correlated in terms of gas and liquid Reynolds numbers. Free-draining experiments were performed to determine the dynamic and static liquid hold-ups. The measured axial dispersion coefficients were higher than those presented in other studies. The dynamic hold-up derived from RTD agreed with total hold-up from free-draining experiments. The static hold-up was found very high, even higher than the dynamic one, due to the liquid accumulated inside the catalyst bed. Possibly, the liquid considered “static” from the viewpoint of the free-draining experiments becomes “dynamic” during the normal column operation.     

The statistical theory of linear capillary chromatography with uniform stationary phase

Based on the random walk model and probability theory, general relations between the moments of column residence time and the moments of step sojourn time and step displacement are established. And starting from the mass-balances principle of solute molecules in the mobile and stationary phases, the moments of step sojourn time and step displacement are derived and expressed in terms of the basic parameters. Substituting the step moments into the general relations, the moments of column residence time are then obtained. The expression of retention time is completely identical to the well-known, the expressions of second moment or HETP unite and generalize various expressions of stochastic theory and mass balance theory, and the third and forth moments are given in more exact form.     

Statistical theory of linear adsorption capillary chromatography with porous-layer stationary phase

A set of accurate expressions of elution-curve moments are derived from the moments of residence time and displacement in a step based on probability theory. Then the problems about residence time and displacement in a step of a solute molecule in the porous layer of capillary columns and in the moving mobile phase are described by a set of mass-balance equations respectively. The set of equations are solved in Fourier-Laplace domain, and the characteristic functions of residence time of a step, as well as the moments, are obtained by means of computing software Mathematica. At last, using numerical inverse Laplace transform, the elution curves for various conditions are calculated. In the case of large desorption constant the results entirely coincide with those of mass-balance-equation theory and in the case of small desorption constant they are equivalent to those of stochastic theory.     

Comparison between the loading capacities of columns packed with partially and totally porous fine particles. What is the effective surface area available for adsorption?

The adsorption isotherms of phenol, caffeine, insulin, and lysozyme were measured on two C(18)-bonded silica columns. The first one was packed with classical totally porous particles (3 microm Luna(2)-C(18)from Phenomenex, Torrance, CA, USA), the second one with shell particles (2.7 microm Halo-C(18) from Advanced Materials Technology, Wilmington, DE, USA). The measurements were made at room temperature (T=295+/-1K), using mainly frontal analysis (FA) and also elution by characteristic points (FACP) when necessary. The adsorption energy distributions (AEDs) were estimated by the iterative numerical expectation-maximization (EM) procedure and served to justify the choice of the best adsorption isotherm model for each compound. The best isotherm parameters were derived from either the best fit of the experimental data to a multi-Langmuir isotherm model (MLRA) or from the AED results (equilibrium constants and saturation capacities), when the convergence of the EM program was achieved. The experiments show than the loading capacity of the Luna column is more than twice that of the Halo column for low-molecular-weight compounds. This result was expected; it is in good agreement with the values of the accessible surface area of these two materials, which were calculated from the pore size volume distributions. The pore size volume distributions are validated by the excellent agreement between the calculated and measured exclusion volumes of polystyrene standards by inverse size exclusion chromatography (ISEC). In contrast, the loading capacity ratio of the two columns is 1.5 or less with insulin and lysozyme. This is due to a significant exclusion of these two proteins from the internal pore volumes of the two packing materials. This result raises the problem of the determination of the effective surface area of the packing material, particularly in the case of proteins. This area is about 40 and 30% of the total surface area for insulin and for lysozyme, respectively, based on the pore size volume distribution validated by the ISEC method. The ISEC experiments showed that the largest and the smallest mesopores have rather a cylindrical and a spherical shape, respectively, for both packing materials.     

A drying step in the protocol to pack capillary columns by centripetal forces for capillary electrochromatography.

Capillary columns have been packed for capillary electrochromatography (CEC) using centripetal forces. The packed columns were maintained under wet conditions or they were dried with nitrogen gas prior to forming the retaining frits. Upon fabrication of the retaining frits, the dried columns were resolvated with the mobile phase. The performance of the columns was evaluated to determine the effect of the drying step during the packing procedure. The columns submitted to the drying step showed improved separation efficiencies and stronger retention characteristics than those kept under wet conditions. The drying step allows the silica-based packing material to be better accommodated inside the capillary column. Upon solvation, the packing material "swells," resulting in a greater packing density, which allows for a stronger retention and improved separation efficiencies. The drying step led to a 13% increase in retention on columns packed with isopropanol. An increase of 15-20% in theoretical plates for the most retained compounds was also observed in such columns.     

Enantioseparation of amino acid derivatives on an immobilized network polymer derived from L-tartaric acid

Seven structurally related amino acid derivatives were successfully enantioseparated by HPLC with a commercially available column containing a chiral immobilized network polymer derived from L-tartaric acid. The experiments were carried out under normal-phase conditions. All the solutes could be baseline separated using n-hexane/2-propanol (95/5) as eluent at a flow rate of 1 ml/min at 25 degrees C, with reasonable retention time (<12 min). The effects of the polar alcohol modifier (type and content) in the mobile phase and the column temperature on the enantioseparation were studied. Apparent thermodynamic parameters were also calculated from the plots of ln alpha or ln k' versus 1/T. Some mechanistic aspects of chiral recognition were discussed with respect to the structures of the solutes. It was found that the enantioseparations are all enthalpy driven, and the N-acyl groups of the solutes have significant influence on the chiral recognition.     

Electronic transition dipole moment directions of reduced anionic flavin in stretched poly(vinyl alcohol) films

The IR and UV/vis linear dichroic spectra of reduced anionic flavin mononucleotide (FMNH-) partially oriented in poly(vinyl alcohol) (PVA) films have been measured to determine the direction of the major electronic transition dipole moments. The IR linear dichroism (LD) was measured in the 1750-1350 cm(-1) region to provide the overall molecular orientation of the FMNH- in the stretched films. Time-dependent density functional theory using the B3LYP functional was used to calculate the normal modes and the transition dipole moments of reduced lumiflavin. The calculated normal modes assisted in IR band assignments and in the determination of the IR transition dipole moment directions which were required for the determination of the orientation parameters for FMNH- in PVA films. The UV/vis LD spectrum was measured over the 200-700 nm region and was resolved into contributions from three pi-->pi* transitions. The directions of the transitions are 90 degrees+/-4 degrees at 440 nm, 79 degrees+/-4 degrees at 350 nm, and 93 degrees+/-4 degrees at 290 nm with counterclockwise rotations with respect to the N5-N10 axis. Comparison of the calculated and experimentally determined transition dipole moments allowed for refined assignment of the transition dipole moment directions. To our knowledge, this is the first experimental evidence that the 350-450 nm absorption arises from two unique transitions. Remarkably, the two lowest energy transition dipole moments for FMNH- are nearly parallel to those obtained in prior studies for both oxidized and semiquinone flavin.     

Preparative anion-exchange chromatography of soybean trypsin inhibitor: the alternative of column-overload methods.

The objective of preparative separation is to purify the largest amount of material in the shortest time and at a minimum cost, i.e. to maximize throughout. One of the techniques for increasing throughput is to overload the column while maintaining purity and cycle time at the same level. This principle is applied in sample displacement mode chromatography, in which the column is overloaded with sample mixture until it is completely saturated. Soybean trypsin inhibitor was purified from a crude protein extract by this technique using an analytical anion-exchange column with small particle size (20 microns). The comparison of these results, using the criterion of throughput, with those derived from a conventional scale-up, using a 40-microns preparative column, led to the conclusion that the overloaded 20 microns column gave a higher throughput than the 40-microns column.     

一种多通道匀浆装填毛细管色谱柱的新装置及应用

针对目前毛细管色谱柱装柱效率低、不同批次装填的毛细管色谱柱之间性能差异大的问题,我们发展了一种多通道匀浆装填毛细管色谱柱的新装置。该装置以液相色谱泵提供压力、采用磁力搅拌保持匀浆液均匀分散,一次可装填多达6根毛细管色谱柱。以牛血清白蛋白(BSA)的胰蛋白酶酶切肽段混合物为样本,选择峰容量、蛋白覆盖率、3个特定离子的保留时间以及毛细管色谱柱柱压为指标,在毛细管液相色谱-质谱联用系统上对装填的反相毛细管色谱柱的性能进行了评价。分别考察了一次装填的6根毛细管色谱柱、两次装填的12根毛细管色谱柱以及一次装填1根与一次装填6根毛细管色谱柱的性能及稳定性。实验结果表明:同一批次装填的6根毛细管色谱柱的性能相近;不同批次装填的12根毛细管色谱柱的峰容量和覆盖率没有明显的区别,但保留时间和毛细管色谱柱柱压的稳定性较差;一次装填1根和一次装填6根毛细管色谱柱柱性能的稳定性与两次分别装填6根毛细管色谱柱的稳定性相近,即采用本装置可显著提高毛细管色谱柱的装填效率且每次装填毛细管色谱柱的数量不会对柱性能产生影响。     

Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations. The simulations showed the separation depended on both the fluid residence time and the number of theoretical plates. However, when the number of theoretical plates was increased to ≥150, the simulations predicted that the separation performance of the column was not significantly improved. The approach described here could be used as a method to quantify acceptable height equivalent of a theoretical plate values for columns, and serve as a tool to understand how column packing quality impacts a given chromatographic separation prior to column scale-up, as well as during the monitoring of column lifetime in the manufacturing of large scale protein therapeutics.     

Understanding and mitigating conductivity transitions in weak cation exchange chromatography

Large conductivity fluctuations were observed during a high pH wash step in a weak cation exchange chromatography process. These conductivity transitions resulted in a conductivity drop during pH increase and a conductivity rise during pH decrease. In some cases, the absolute conductivity change was greater than 6 mS/cm which was sufficient to affect target protein retention on the column. Further investigation revealed that wash buffer concentration, resin ligand density, and resin ligand pK have a profound effect on the magnitude of the conductivity transitions and the shape of corresponding pH traces. A potentiometric electrode selective for sodium ions was used to measure effluent counterion concentrations from two preparative resins during high pH washes, and the number of exchangeable counterions was compared to predictions made using ion exchange equilibrium theory. Results from this analysis show that conductivity transitions can be effectively mitigated without compromising process performance by optimizing the trade-off between wash buffer concentration and wash phase duration.     

The reproducible acquisition of comparative liquid chromatography/tandem mass spectrometry data from complex biological samples

An in-depth study of the reproducibility of data acquired for comparative proteomics analysis using a prototype two-stage heated laminar flow chamber fitted to a commercial high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) instrument was undertaken. The study is based on 24 replicate samples from four independent membrane preparations derived from two matched breast cancer cell lines. Variation and reproducibility in the data were evaluated at several levels highlighting the relative efficiency and variability of the acquisition routines used. Specifically, variation in the number and relative intensities of chromatographic peaks eluted from the LC column, precursor ion selection and sequence identification were evaluated. On average, approximately 6500 chromatographic peaks were generated for each acquisition with a corresponding coefficient of variance (CV) of less than 20%. Precursor ion selection and sequence identification averaged 1380 and 780 events per acquisition sample, respectively, with corresponding CVs of less than 10% for each. The reproducibility in the precursor ion selection was typically better than 60% between similar replicates. Using protein and peptide internal standards, it was found that the CV in retention time across the gradient between two acquisition pairs was typically less than 5%, whereas the average intensity ratio was 1.0 (expected) with a CV approaching 20%. An evaluation of the intensity ratios calculated from endogenous peptide sequences, identified across the acquisition set, indicated a CV of approximately 30%. Similarly, the CV associated with the top 1000 peptides indicated a mean and median of 28.4 and 26.95%. For a given acquisition pair it was also found that approximately 11% of the chromatographic peaks eluting from the column were linked to a sequence or identified. For these experiments, less than 10% of the peak pairs had absolute ratios greater than 2.0 and of those only approximately 10% had sequences linked to them. For each matched acquisition set on average 406 proteins were identified with a CV of less than 10%. Of the proteins that were identified approximately 30% had at least one predicted trans-membrane domain, indicating a four-fold increase over a crude homogenate sample with only minor enrichment. During these experiments it was found that the interface did not significantly alter the relative charge state distribution of ions, nor did it introduce significant interference from background ions. The interface was capable of 24-hour acquisition cycles.     

Direct determination of residual Pluronic F-68 in in-process samples from monoclonal antibody preparations by high performance liquid chromatography

A simple and sensitive high performance liquid chromatography (HPLC) method was developed to determine residual Pluronic F-68 (PF-68) in in-process samples of monoclonal antibody (MAb) preparations. The method permits the direct injection of proteinaceous samples after simple sample dilution and is able to quantitate as low as 50 mg/L of PF-68 in the presence of up to approximately 30 g/L of protein. The PF-68 molecule was separated on a restricted access reversed phase column using a step gradient and then measured by an evaporative light scattering detector (ELSD). The method was successfully applied to demonstrate PF-68 clearance in MAb purification processes. A modified colorimetric method using liquid-liquid extraction and cobalt thiocyanate to derivatize PF-68 is also described. The results obtained by both the HPLC and colorimetric methods were compared. In addition to its ease of use and simplicity, the HPLC method had better accuracy and higher throughput than the colorimetric method.     

Online extraction and determination of octylglucoside by reversed-phase high-performance liquid chromatography with evaporative light-scattering detection

A reversed-phase high-performance liquid chromatography method using evaporative light-scattering detection is developed for the determination of residual octylglucoside (OG) levels after a detergent exchange step for in-process samples of a vaccine antigen. The reversed-phase column not only provides separation of the OG but also functions as an extraction column to remove the vaccine antigen from the sample, thereby eliminating off-line sample manipulations. In addition to column selection, the mobile phase is optimized to enhance extraction and separation. The vaccine antigen is irreversibly bound to the column, allowing nonprotein components to interact with the column for separation and elution. The assay is linear over the range of 0.00050-0.050% OG. Precision tested at 0.0010% and 0.0050% OG is 2.9% and 7.2% relative standard deviation, respectively. The limits of quantitation and detection are determined to be 0.00050 and 0.000125% OG, respectively. Accuracy is determined to be 103 and 98%, based on spike recoveries of 0.0010% and 0.0050% OG, respectively.     

External mass transfer in high performance liquid chromatography systems

External mass transfer in a HPLC system operated in the reversed-phase mode was studied by pulse response experiments, using a column packed with non-porous C(18)-silica gel spherical particles, 18 microm in diameter. The first and second moments of the elution peaks, measured under different flow velocities and temperatures, were analyzed by the moment method to determine the external mass transfer coefficient (k(f)). The dependence of the Sherwood number on the Reynolds and the Schmidt numbers is almost the same as that observed in previous investigations of conventional literature correlations. The exponent of the last two nondimensional parameters was derived as being in the range from 0.28 to 0.41. When the Kataoka equation is used, the mean square deviation was calculated to be 0.21 for the values of k(f) estimated in this study. It is concluded that conventional correlations can be used to estimate k(f) values, even when the particle diameter is of the order of a micrometer.     

Ab initio study of the electronic states of BS including spin–orbit coupling

The entire 30 Ω states generated from the 12 valence and two Rydberg Λ–S states of the BS radical have been studied at the MR-CISD + Q level of theory for the first time. The effects of spin–orbit coupling and the avoided crossing rule between electronic states of the same symmetry were analyzed. Spectroscopic constants of several excited states that have never been observed in experiment were obtained. The transition properties of several low-lying bound excited states to ground state transitions, including the transition dipole moments and the Franck–Condon factors, were also calculated, from which the corresponding single-channel radiative lifetimes were derived.     

Statistical theory of multiple-site linear wall-adsorption capillary Chromatography

Based on the mass-balance principle, a particular diffusion equation to describe the movement of solute molecules in the stagnant layer of multiple-site solid surfaces is constructed. From the equation, the moments of residence time in a step on multiple-site surfaces are derived. Similarly, the moments in a step in the mobile phase are also derived from a diffusion-drift equation. According to the probability theory, there exists a general relationship between the moments of an elution curve and the moments in a step. Through this relationship, the expressions of the elution-curve moments are derived from the step moments. In this paper, the details related to multiple-site linear wall-adsorption capillary chromatography are described and added in the equations to determine the step moments. The resultant expressions of the elution-curve moments involve various factors, such as adsorption–desorption rate constants, equilibrium constants, axial and radial dispersions in the mobile phase. Afterwards, the moment expressions are used to analyze the peak tailing. The results show that a small quantity of sites with a slow desorption rate will lead to a large peak asymmetry.     

Revisiting the photophysical properties and excited singlet-state dipole moments of several coumarin derivatives

The solvent effects on the electronic absorption and fluorescence emission spectra of several coumarins derivatives, containing amino, N,N-dimethyl-amino, N,N-diethyl-amino, hydroxyl, methyl, carboxyl, or halogen substituents at the positions 7, 4, or 3, were investigated in eight solvents with various polarities. The first excited singlet-state dipole moments of these coumarins were determined by various solvatochromic methods, using the theoretical ground-state dipole moments which were calculated by the AM1 method. The first excited singlet-state dipole moment values were obtained by the Bakhshiev, Kawski-Chamma-Viallet, Lippert-Mataga, and Reichardt-Dimroth equations, and were compared to the ground-state dipole moments. In all cases, the dipole moments were found to be higher in the excited singlet-state than in the ground state because of the different electron densities in both states. The red-shifts of the absorption and fluorescence emission bands, observed for most compounds upon increasing the solvent polarity, indicated that the electronic transitions were of π-π* nature.     

填充条件对半制备柱性能影响的机理研究

对稀匀浆法填充半制备柱(Ф10 mm×100 mm)的过程进行研究,并用正交法对柱填充条件进行了优化,考察了压力、匀浆液组成、匀浆液体积对半制备柱填充效果的影响.由柱效和分离度定义式中的参数入手,讨论填充条件对柱性能的影响.通过计算总孔隙率ε_t、内部孔隙率ε_p、外部孔隙率ε_e、Van-Deemter曲线、吸附等温线等参数,发现填充压力对填充密度的作用最大,是最重要的影响因素;匀浆配比和匀浆体积共同影响其填充的均匀性.在压力11 MPa,流速275 mL/min,120 mL纯乙醇为匀浆液的条件下得到了最好的填充效果.     

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。