Chromatographic behaviour of low molar-mass polyesters in normal-phase high-performance liquid chromatography

Summary The normal-phase chromatographic retention behaviour of polyesters on bare silica and on a polymer-based polyamine (PA) column has been studied with a variely of binary mobile phases under isocratic conditions. The dependence of experimental retention data on the degree of polymerization (p) and on mobile phase composition (φ) was characterized by to an approach developed by Jandera et al. The bulky repeating unit and the relatively highly polar end groups of the polyesters both had a large influence on retention behaviour. The two effects in combination explain the molar-mass-independent retention observed experimentally at a particular mobile phase composition for all the mobile phase—stationary phase combinations investigated. These conditions were found to be independent of the type of end group. End group separation on a silica column improves when the polarity of the less polar solvent is increased. End group separation is better on the PA column because of a greater difference between the adsorption energy of the alcohol and acid end groups. Better prediction of retention data on the PA column was achieved by use of an approach which assumes two different types of adsorption site. Results enabled further understanding of retention behaviour in normalphase gradient polymer-elution chromatography (NPGPEC) and explained both the dependence of the order of elution onp and differences between the end-group selectivity of different systems.     

Characterization of polystyrene and polyisoprene by normal-phase temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) is applied to the characterization of polyisoprene (PI) and polystyrene (PS) using normal-phase (NP) stationary phase--bare silica or diol bonded silica. Tetrahydrofuran-isooctane mixtures are used as a mobile phase. PI and linear and star shaped PS samples are successfully fractionated in terms of the molecular mass with a high resolution comparable to that of reversed-phase (RP) HPLC. Temperature dependence of the retention shows that the enthalpy of adsorption of PS to the stationary phase is exothermic. In addition, some characteristic features of the NP-TGIC system relative to those of RP-TGIC are presented, which include a high sensitivity on the polar end group and the simultaneous size-exclusion chromatographic and TGIC characterization of PS and PI mixtures.     

Effect of pore size of packing materials on molecular-weight separation by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) is an interactive polymer chromatography technique varying the column temperature during the elution in a programmed manner to control the solute retention. In the present paper, the effect of the pore size of packing materials on the molecular-weight separation of polystyrene and poly(methyl methacrylate) standard samples by TGIC was studied by using the columns (octadecyl modified silica) with different pore size (100, 300 and 1000 Å) and eluent mixture of CH₂Cl₂/CH₃CN. By rising temperature gradient, both polymers were separated by molecular weight from lower to higher. It became clear that each sample elutes out earlier as the pore size is larger. These experimental results could be explained by the theory based on the scaling concept of Gorbunov and Skvortsov.     

Effect of temperature on DNA fractionation in slalom chromatography

Slalom chromatography is an alternative chromatographic procedure for the analysis of relatively large double-stranded DNA molecules and is based on a hydrodynamic principle. The retardation of the DNA fragments from the cleavage of the Lambda DNA by the KpnI restriction enzyme was studied using an acetonitrile-phosphate buffer as a mobile phase (flow rate equal to 0.3 ml/min) and a C1 column as a stationary phase at various temperatures. It was shown that the temperature constituted an important parameter for the separation of the DNA fragments in slalom chromatography. The DNA hydrodynamic behavior with the temperature was related to the variation in the fluid viscosity and the modification of the elastic properties of the biopolyrner.     

Characterization of a 4-miktoarm star copolymer of the (PS-b-PI)3 PS type by temperature gradient interaction chromatography

Temperature gradient interaction chromatography (TGIC) was applied for the separation of a complex miktoarm star copolymer which has one polystyrene (PS) arm and three polystyrene-b-polyisoprene (PS-b-PI) diblock copolymer arms. Such miktoarm star polymers are much more difficult to characterize than branched homopolymers since the byproduct, typically polymers with missing arm(s) or coupled products, have not only different molecular weights but also different compositions. TGIC was able to fully separate the byproducts, and the composition of the molecular species corresponding to the different separated elution peaks was determined by two methods, fractionation/NMR and multiple detection (UV and RI). A reasonable agreement between the results of the two methods was obtained. By using the composition found, the corresponding molecular weights were determined by multi-angle light scattering detection. Based on the composition and the molecular weight we were able to identify the structure of the different molecular species.     

Axial temperature gradient and mobile phase gradient in microcolumn high-performance liquid chromatography

The effect of axial temperature gradient (ATG) along a microcolumn on the separation performance at both isocratic and gradient elution mode was investigated. A thermostat system was designed to form an ATG along the packed column. Polycyclic aromatic hydrocarbons (PAHs) were separated on a 0.53 mm  × 150 mm i.d. 5 μm C₁₈ microcolumn, with water and acetonitrile as mobile phase. The separation results obtained at mobile phase gradient (MPG) and ATG in microcolumn HPLC were compared with the results performed at ambient conditions. Extrapolated curves of peak width at half height (wh)versus lnk showed that wh is narrower at the same retention time when ATG was applied in addition to MPG. The column efficiency was enhanced 20-30% and the resolution was slightly reduced because of reduction of selectivity at elevated temperature at ATG condition. The RSD of retention time in ATG mode was less than 2.5%.     

Turbidimetric titration and gradient high-performance liquid chromatography of polystyrene samples

Summary Polystyrene samples of narrow molecular-weight distribution have been eluted according to their molecular weight from columns packed with bare silica Si50, phenyl, or C18 bonded phase by gradients of methanol and tetrahydrofuran (THF) or ofiso-octane and THF. Among the six combinations investigated,iso-octane/THF with a silica column formed a proper normal-phase system whereas methanol/THF with a C18 column formed a proper reversed-phase system. The combinations of C18 column andiso-octane/THF or of Si50 column and methanol/THF gradient did not correspond to the approved polarity rules in high-performance liquid chromatography but were nevertheless effective in separating polystyrene mixtures by molecular weight. Methanol andiso-octane are nonsolvents for polystyrene whereas THF is a solvent. The solubility of polystyrene as a function of molecular weight and concentration was determined by means of turbidimetric titration of solutions in THF with the nonsolvents used in the gradients. The solubility and elution characteristics were almost identical on C18 columns or in methanol/THF combinations. The elution from phenyl bonded phase and Si50 columns usingiso-octane/THF gradients required more THF than the solubility experiments. Information is also given on the occurrence of multimodal elution patterns.     

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。     

Indirect detection in reversed-phase liquid chromatography: Ion-pair retention models for cations on polystyrene polymers

Summary The distribution equilibria of cationic compounds in reversed-phase chromatographic systems (ion-pair chromatography) have been studied on the basis of their effect on a detectable mobile phase component. The solid phase was a polystyrene-divinylbenzene copolymer and the detectable component, a quaternary ammonium ion, 1-methylpyridine. The solutes were mono- and divalent amines and quaternary ammonium ions. The cations can be retained by ion-pair adsorption and ion exchange. Expressions for the ion-pair retention of the solutes and the mobile phase cation (system peak) have been developed assuming Langmuir distribution of ion pairs to a solid phase with one kind of binding site. The validity of the expressions has been tested by evaluation of ion-pair distribution constants using non-linear curve fitting techniques. Good agreement for the constants of common ion pairs was obtained from different kinds of capacity ratio expressions. Ion exchange retention can appear beside ion-pair retention, and it has been observed in the pH range 1.6–6.1. The effect depends not only on cations in the mobile phase, but also on the nature of the buffering systems.     

Preparation of hydrophilic polymer-grafted polystyrene beads for hydrophilic interaction chromatography via surface-initiated atom transfer radical polymerization

A one-step procedure based on surface-initiated atom transfer radical polymerization (SI-ATRP) to hydrophilize monodisperse poly(chloromethylstyrene-co-divinylbenzene) beads has been presented in this work, using 2-hydroxyl-3-[4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl]propyl 2-methylacrylate (HTMA) as a monomer. The chain length of the grafted poly(HTMA) was controlled via varying the ratio of HTMA to initiator on the surface of the beads. When using the grafted beads as a stationary phase in hydrophilic interaction chromatography (HILIC), good resolution for nucleobases/nucleosides was obtained with acetonitrile aqueous solution as an eluent; while for phenolic acids and glycosides, they could be eluted and separated in the presence of TFA. The retention time of the solutes increased with the amount of the grafted HTMA. The retention mechanisms of solutes were investigated by the effects of mobile phase composition and buffer pH on the retention of solutes. The results illustrated that the retention behaviors of the tested solutes were dominated by hydrogen bonding interaction and electrostatic interaction. From the chemical structure of the ligands, the modified beads could not only be used as a stationary phase in HILIC, but also act as a useful building block to develop new stationary phases for other chromatographic modes such as affinity media.     

The temperature dependence of retention indices in gas chromatography

Summary A linear dependence of (T–T₁)/[1(T)–1(T₁)] on temperature (considering the retention index 1(T₁) at temperature T₁ as a standard value) is derived. Both ther retention index at an assigned temperature and the temperature dependence of the retention index can be calculated from retention data measured at two temperature-programing rates.     

A data base for polymer chromatography: Temperature dependence of interaction parameters in liquid adsorption chromatography

The temperature dependence of retention behaviour of polyethylene glycol (PEG) and its mono‐ and dimethyl ethers was studied on various RP columns in different mobile phases. The accessible volumes and the interaction parameters were determined from slope and intercept in a plot of the elution volumes of the oligomers of a polymer homologous series as a function of the difference of the elution volumes of consecutive oligomers. A quite different dependence of the interaction parameters was observed in the different mobile phases. While in methanol–water the interaction parameter decreases with increasing temperature, the opposite effect is observed in acetonitrile (ACN)–water. In acetone–water, the temperature dependence is almost negligible.     

Recombinant protein purification using gradient assisted simulated moving bed hydrophobic interaction chromatography. Part II: process design and experimental validation

In the first part of this work adsorption isotherm parameters were acquired to describe the migration of recombinant streptokinase in Butyl Sepharose columns at different salt concentrations. Based on these results, a simulated moving bed (SMB) chromatographic process was designed and realised, which exploits a two-step salt gradient and allows the continuous separation of streptokinase from contaminants present in a clarified Escherichia coli cell lysate solution. This second part describes the design of the three-zone open-loop gradient SMB process applying both equilibrium theory and an equilibrium stage model and presents results of a series of experiments aiming to obtain pure streptokinase. Moreover, the potential of the SMB process and the design approach are evaluated.     

Two-dimensional liquid chromatography analysis of synthetic polymers using fast size exclusion chromatography at high column temperature

In recent years, two-dimensional liquid chromatography (2D-LC) has been used increasingly for the analysis of synthetic polymers. A 2D-LC analysis provides richer information than a single chromatography analysis at the cost of longer analysis time. The time required for a comprehensive 2D-LC analysis is essentially proportional to the analysis time of the second dimension separation. Many of 2D-LC analyses of synthetic polymers have employed size exclusion chromatography (SEC) for the second-dimension analysis due to the relatively short analysis time in addition to the wide use in the polymer analysis. Nonetheless, short SEC columns are often used for 2D-LC analyses to reduce the separation time, which inevitably deteriorates the resolution. In this study, we demonstrated that high temperature SEC can be employed as an efficient second-LC in the 2D-LC separation of synthetic polymers. By virtue of high temperature operation (low solvent viscosity and high diffusivity of the polymer molecules), a normal length SEC column can be used at high flow rate with little loss in resolution.     

糖类化合物亲水作用色谱保留行为评价

以糖类化合物为研究对象,系统评价了其在亲水模式下的色谱保留行为。分别考察了流动相、固定相和缓冲盐等对糖类化合物保留的影响,建立了糖类化合物在亲水模式下的保留方程。结果表明,糖类化合物随着流动相中乙腈比例的降低,保留时间减小;随着缓冲盐浓度的增加,保留时间增加;同时,糖类化合物的保留行为还会受到有机溶剂种类和固定相类型的影响;其保留行为可使用顶替吸附-液相相互作用模型定量描述。将该模型进一步用于实际样品中糖类化合物保留行为的预测,获得了较好的实验结果,预测保留时间与实测保留时间的相对误差小于0.3%。对糖类化合物亲水模式下的保留行为进行了系统的评价和定量描述,该研究结果将有助于糖类化合物亲水作用色谱分离方法的发展。     

Optimization strategies in micellar electrokinetic capillary chromatography. Optimization of the temperature of the separation capillary

Summary The influence of the temperature of the capillary on the separation of small molecules by micellar electrokinetic chromatography with buffers containing sodium dodecyl sulfate was examined. Investigated parameters were the electroosmotic velocity, the migration velocity of the micelles, the strength of the electric current, retention factors and plate numbers. Thermodynamic parameters associated with micelle solubilization were evaluated from the variation of the retention factor with temperature. An investigation was carried out as to whether the dependence of the electroosmotic velocity and the migration velocity of the micelles are mainly due to the decrease in viscosity and dielectric constant with increase in temperature. The temperature of the capillary strongly affects the plate numbers for various solutes obtained with the same chromatographic system. In order to exclude secondary effects by alteration of the instrumental band broadening and the electroosmotic velocity with temperature, a method is presented that permits the development of chromatograms in MEKC at various temperatures with constant injected plug length and constant electroosmotic velocity. The results are discussed from the point of view of minimizing the analysis time and optimizing the resolution.     

Optimization of stepwise gradient elution in columns chromatography

Summary A general equation for the final retention of a solute chromatographed under conditions of stepwise gradient elution has been derived. The elution process and the distances travelled by solutes as a function of eluent volume were simulated by computer for the optimization of stepwise gradient prorams from isocratic HPLC data. The validity of the equations was experimentally veritied.     

Comparison of normal-phase and reversed-phase gravity-flow column methods for provitamin A determination

Summary Considering the high cost of HPLC, the use of preparative C₁₈ packing material as stationary phase in classical gravity-flow columns is proposed as an alternative for developing countries. In the present study, three methods were compared, one utilizing a normal-phase MgO: HyfloSupercel column, developed with increasing concentration of acetone in petroleum ether and two utilizing C₁₈ reversed-phase columns developed with acetonitrile-methanol-chloroform (RP-C₁₈ method) or with decreasing concentration of water in acetone (modified RP-C₁₈ method). Percent recoveries of -carotene from the columns were 98, 96 and 98% and of the entire analysis (-carotene added to kale) 92, 89 and 92%, for the normal phase, RP-C₁₈ and modified RP-C₁₈ methods respectively. No significant difference was observed in the provitamin A contents (exclusively -carotene) of two leafy vegetables determined by the modified reversed-phase and normal-phase methods; however, significantly lower values were obtained by the RP-C₁₈ method. The situation became more complicated when samples containing other provitamins were analyzed. For squash both types of stationary phase could separate - and -carotene; however, for tomato and red-fleshed papaya, part of lycopene remained mixed with -carotene in both reversed-phase columns. Although -cryptoxanthin was separated in the modified RP column, it was also mixed with lycopene in the RP column. For all samples, the normal-phase column demonstrated much better separation.     

顶空气相色谱法测定聚苯乙烯日用品中可溶性苯乙烯单体

应用带有顶空进样器气相色谱仪,采用外标法定量,分别以乙醇、乙醇 水作提取剂,分析了聚苯乙烯日用品中可溶性苯乙烯的含量,并对其提取温度、时间进行了研究。在选择的色谱条件下,用乙醇、乙醇 水提取苯乙烯标准溶液分别在0.8～5000mg L、0.1～200mg L浓度间呈现良好的线性关系,检出限<0.1mg L,其相对标准偏差分别为3.2%、5.9%。