A novel multi-column gas chromatographic system

Constructional and operating details are given of a special valve permitting selection of any one of four columns for connection to the detection system of a gas chromatograph, all four columns being connected to the carrier gas stream. If the retention times permit it, the columns may be used simultaneously for chromatographic separations and switched in turn into series with the detector for determination of the eluted components.     

A chromatographic response function for automated optimization of separations

Summary A chromatographic response function is studied that is sensitive to important criteria of analytical performance: selectivity, accuracy, precision and time required to perform the separation. This function can be applied to mixtures of unknown solutes and little computational effort is needed to evaluate the numerical value of the function from the chromatogram. It shows well defined optima at elution strengths that give minimal elution times simultaneously with good separations in high-performance liquid chromatography. No prior chromatographic information characterizing the expected performance of a system is required. It is possible to use this function without a mechanistic model describing the separation process.     

Protein separations with induced pH gradients using cation-exchange chromatographic columns containing weak acid groups

The behavior of chromatographic columns packed with resins containing both weak and strong cation-exchange groups is investigated in order to obtain protein separations by means of internally generated pH gradients in response to step changes in buffer composition. A local equilibrium model is developed to predict pH transitions using non-adsorbed buffers, i.e. containing neutral and negatively charged buffering species, based exclusively on the resin titration curve. In agreement with experimental results, the model predicts practical, fairly linear gradients between pH 5 and 7, which are formed using suitable mixtures of acetate and phosphate buffers. The separation of mixtures of ovalbumin, albumin, and transferrin is used as a model system, but, unlike most previous work, we consider preparative conditions. Near baseline resolution is obtained with protein loads as high as 10mg/mL and mobile phase velocities at high as 460 cm/h using porous, 70-microm diameter particles. The peaks obtained with this approach are much sharper than could be obtained isocratically or using externally generated, unretained gradients as a result of the peak compression caused by the axial pH gradient formed along the column. Moreover, separation is obtained at very low ionic strengths (2-3 mS/cm). The effects of flow velocity, mobile phase composition, time of injection, and protein load on retention and elution pH are investigated systematically demonstrating a range of ways in which the separation can be controlled and optimized.     

Monolithic continuous beds as a new generation of stationary phase for chromatographic and electro-driven separations

Monolithic supports are a new generation of stationary phases which are typically prepared using a simple molding process carried out within the confines of the capillary. They provide high rates of mass transfer at lower pressure drops, enable much faster separations and the nature of the pores allows easy permeability for large molecules. This review summarizes the current achievements and application of organic polymer-based monolithic columns, silica-based monolithic columns and their application in bioaffinity processing, modern biotechnology, determination of microorganisms and chiral separations. Special attention is paid to microfabricated devices with monolithic supports because their fabrication of particles directly in the channel eliminates the need for a frit and also creates a unique homogeneity of packing.     

Synthesis of zirconia monoliths for chromatographic separations

The aim of this work is to join the advantages of two different kinds of stationary phases: monolithic columns and zirconia-based supports. On the one hand, silica monolithic columns allow a higher efficiency with a lower back-pressure than traditional packed columns. On the other hand, chromatographic stationary phases based on zirconia have a higher thermal and chemical stability and specific surface properties. Combining these advantages, a zirconia monolith with a macroporous framework could be a real improvement in separation sciences. Two main strategies can be used in order to obtain a zirconia surface on a monolithic skeleton: coating or direct synthesis. The coverage by a zirconia layer of the surface of a silica-based monolith can be performed using the chemical properties of the silanol surface groups. We realized this coverage using zirconium alkoxide and we further grafted n-dodecyl groups using phosphate derivatives. Any loss of efficiency was observed and fast separations have been achieved. The main advance reported in this paper is related to the preparation of zirconia monoliths by a sol-gel process starting from zirconium alkoxide. The synthesis parameters (hydrolysis ratio, porogen type, precursor concentration, drying step, etc.) were defined in order to produce a macroporous zirconia monoliths usable in separation techniques. We produced various homogeneous structures: zirconia rod 2 cm long with a diameter of 2.3 mm, and zirconia monolith inside fused silica capillaries with a 75 microm I.D. These monoliths have a skeleton size of 2 microm and have an average through pore size of 6 microm. Several separations have been reported.     

Monolithic silica columns for high-efficiency chromatographic separations

A deconvolution methodology for overlapped chromatographic signals is proposed. Several single-wavelength chromatograms of binary mixtures, obtained in different runs at diverse concentration ratios of the individual components, were simultaneously processed (multi-batch approach), after being arranged as two-way data. The chromatograms were modelled as linear combinations of forced peak profiles according to a polynomially modified Gaussian equation. The fitting was performed with a previously reported hybrid genetic algorithm with local search, leaving all model parameters free. The approach yielded more accurate solutions than those found when each experimental chromatogram was fitted independently to the peak model (single-batch approach). The improvement was especially significant for those chromatograms where the peaks were severely affected by the tails of the preceding compounds. Peak shifts among chromatograms, which are a usual source of non-bilinearity, were modelled in a continuous domain instead of in a discrete way, which avoided some drawbacks associated with latent variable methods. An experimental design involving simulated chromatograms was applied to check the method performance. Five main factors affecting the deconvolution were examined: concentration pattern, chromatographic resolution, number of batches and replicates, and noise level, which were evaluated using first- and second-order figures of merit. The method was also tested on three real samples containing compounds showing different overlap. Four multi-batch deconvolution methods were considered differing in the nature of the processed information and kind of peak matching among chromatograms. In all cases, the multi-batch deconvolution yielded better performance than the single-batch approach.     

Improved thin-layer chromatographic method for sugar separations

A method is described for the utilization of pre-coated, non-impregnated silical gel thin layers in one- and two-dimensional separations of carbodhyrates and related compounds. Boric and phenylboronic acids were added to the organic elution systems in different concentrations and their interactions with the sugar molecules during the chromatographic process were studied. A comparison was made between solvent systems containing boric or phenylboronic acid and systems devoid of both acids as eluents. With boric acid-containing solvents the migration of some sugars was considerably inhibited, whereas phenylboronic acid produced an increase in the RF values of certain sugars. The combination of these two types of solvent in two dimensional development resulted in the clear separation of a group of mono- and disaccharides of bioclinical interest.     

Estimating optimal time for fast chromatographic separations

The term t_{min cc} provides a ready estimate of the shortest time that can be obtained by “column cutting” for baseline resolution of two components showing excess chromatographic resolution. While actual column cutting is impractical, the t_{min cc} value is shown to be closely related to the minimum separation time obtainable by adjusting other parameters such as flow rate, mobile phase composition, and temperature, affording scientists interested in the development of fast chromatographic separations a convenient tool for estimating the minimum separation time that can be obtained by modifying a given method development screening result. Furthermore, the relationship between t_{min cc} and the minimum separation time obtainable by adjusting other parameters is shown to be dependent on the speed of the screening method, with aggressive screening gradients affording t_{min cc} estimates that match the actual minimum separation time, and “lazy” screening gradients affording t_{min cc} values that overestimate minimum separation time. Consequently, the analysis of the relationship between t_{min cc} and actual minimum separation time may be a useful tool for determining the “fitness” of method development screening methods.     

基于集成化多柱二维液相色谱系统分析血清中的氨磺必利

快速准确的治疗药物监测对于临床上确保患者用药有效性及安全性至关重要,同时也能够确定患者用药依从性,制定个性化给药方案。该文以两支疏水性略有差异的反相分离柱Supersil ODS2和SinoChrom ODS-BP,及强阳离子交换捕集柱Supersil SCX构建了基于集成化的多柱二维液相色谱系统。通过二维色谱接口,以pH 3.0的磷酸缓冲液调整第一维分离后的洗脱液组成,降低有机相含量并维持pH,改善了中心切割模式下样品转移和捕集的效率。利用该多柱二维液相色谱系统发展了血清中氨磺必利的二维液相色谱检测方法,血清样品经过高氯酸和甲醇混合液沉淀蛋白质并离心后直接300 μL大体积进样,以乙腈/磷酸缓冲液(25 mmol/L, pH 3.0)(20/80, v/v)作为第一维分离流动相,磷酸缓冲液(25 mmol/L, pH 3.0)作为捕集过程的稀释流动相,乙腈/磷酸缓冲液(25 mmol/L, pH 7.0)(25/75, v/v)作为第二维分离流动相,12 min内即可完成分析。方法在10~200 ng/mL的范围内线性相关性良好(r=0.9998)。样品在50 ng/mL和100 ng/mL两个加标浓度下的回收率稳定,在73.7%~76.8%之间。方法的检出限为7.28 ng/mL,定量限为24.27 ng/mL,能够满足《神经精神药理学治疗药物检测共识指南》中推荐的药物监控范围要求。由于该系统日常使用及维护成本较低,且能够实现自动化分析,故该方法适合在临床上用于治疗药物监测研究。     

A novel pseudo simulated moving bed with solvent gradient for ternary separations

A novel pseudo simulated moving bed was suggested to separate a ternary mixture. A solvent gradient was created to make the solvent strength decreasing from zone II to zone III. Under suitable conditions, the least retained solute A moved forward and the most retained solute C moved backward in zones II and III whereas the medium retained solute B moved forward in zone II but backward in zone III to be trapped in the two zones consequently. Once the columns in zones II and III were saturated with solute B, the solvent dissolving the feed was introduced at the feed port to remove solute A from the raffinate-port and solute C from the extract-port. Finally, solute B was recovered from the extract port by stopping the liquid flow in zone II. This scheme was validated by the successful separation of dihydrocapsaicin from capsaicinoids.     

Chiral metal-organic frameworks for high-resolution gas chromatographic separations

Chiral metal-organic framework coated open tubular columns are used in the high-resolution gas chromatographic separation of chiral compounds. The columns have excellent selectivity and also possess good recognition ability toward a wide range of organic compounds such as alkanes, alcohols, and isomers.     

Sample preparation for chromatographic separations: an overview

Chromatographic methods dominate the field of organic analysis. However, many samples are too dilute, too complex or incompatible with the chromatographic system to render separation or detection possible without preliminary matrix simplification and/or preconcentration. A broad selection of the most common, recent and emerging isolation, clean-up and concentration techniques used to prepare samples for chromatographic separations are reviewed.     

Development of fluorinated,monolithic columns for improved chromatographic separations of fluorous-tagged analytes

With applications that take advantage of highly selective fluorine–fluorine interactions appearing with greater frequency in the literature, the development of porous polymer monoliths from fluorous components is reported here for the purpose of chromatography of tagged analytes. With potential uses in fields as diverse as separation science and proteomics, facile fabrication of materials with fluorous specificity that can be applied in a high-throughput manner is greatly desirable. To this end, we have developed porous polymer capillary columns with varied fluorous content using a simple UV-initiated radical polymerization process and characterized them using flow-induced backpressure and scanning electron microscopy (SEM). With structural similarities assured (visually, and by backpressure variations of less than 42%), the monoliths were tested as chromatographic columns for the separation of a series of fluorous-tagged analytes under gradient conditions. It was found that columns made with fluorinated components exhibited greater selectivity for fluorous analytes than did equivalent, non-fluorinated monoliths, retaining analytes with either one or two fluorous tags for approximately 6% and 13% longer, respectively. This supports the idea of differences existing between fluorous and reverse-phase separation mechanisms, and encourages a broader range of potential applications for fluorous monoliths of this type.     

Continuous stationary phase gradients for planar chromatographic media

A simple, elegant method for the formation of a continuous stationary phase gradient for use in chromatographic separations is described. Its applicability to separation science is demonstrated using thin-layer chromatography as a test case. Gradient stationary phases were formed on activated High Performance Thin-Layer Chromatography (HP-TLC) plates using a newly developed methodology termed "controlled rate infusion". Specifically, the SiOH groups on the activated HP-TLC plates were reacted with 3-aminopropyltriethoxysilane (APTEOS) in a time dependent fashion by using a programmable syringe pump to control the rate of APTEOS infusion into the deposition reservoir. The shape (profile) of the gradient was controlled by the rate of infusion and imaged by taking advantage of the concentration-dependent color formation reaction between amine groups and ninhydrin. The advantages of such gradients in optimizing the retention and separation of various components in different mixtures were illustrated using mixtures of (1) four weak acids and bases and (2) three widely used over-the-counter drugs. The separation of the individual components on the gradient stationary phase was clearly improved relative to those on either traditional normal-phase TLC plates or uniformly amine-modified TLC plates. Precise control over component retention and separation was also demonstrated by strategically modifying the steepness of the gradient.     

Use of 120-nm deep channels for liquid chromatographic separations

The present study reports on the exploration of the separation speed limits of RPLC chromatography in open-tubular channels. Applying the shear-driven chromatography principle in a 120-nm deep open channel, and using an improved detection set-up, the separation of three coumarin dyes was detectable 8mm downstream of the injection point. At this distance, separation efficiencies of N = 17,900 - 24,100 plates were obtained at a velocity of 10 mm s(-1), corresponding to a plate generation velocity of 21,100 to 28,300 plates per second for the most and least retained component, respectively.     

Fundamental characteristics of synthetic adsorbents intended for industrial chromatographic separations

With the aim of obtaining comprehensive information on the selection of synthetic adsorbents for industrial applications, effect of pore and chemical structure of industrial-grade synthetic adsorbents on adsorption capacity of several pharmaceutical compounds was investigated. For relatively low molecular mass compounds, such as cephalexin, berberine chloride and tetracycline hydrochloride, surface area per unit volume of polystyrenic adsorbents dominated the equilibrium adsorption capacity. On the contrary, effect of pore size of the polystyrenic adsorbents on the equilibrium adsorption capacity was observed for relatively high molecular mass compounds, such as rifampicin, Vitamin B12 and insulin. Polystyrenic adsorbent with high surface area and small pore size showed small adsorption capacity for relatively high molecular mass compounds, whereas polystyrenic adsorbent with relatively small surface area but with large pore size showed large adsorption capacity. Effect of chemical structure on the equilibrium adsorption capacity of several pharmaceutical compounds was also studied among polystyrenic, modified polystyrenic and polymethacrylic adsorbents. The modified polystyrenic adsorbent showed larger adsorption capacity for all compounds tested in this study due to enhanced hydrophobicity. The polymethacrylic adsorbent possessed high adsorption capacity for rifampicin and insulin, but it showed lower adsorption capacity for the other compounds studied. This result may be attributed to hydrogen bonding playing major role for the adsorption of compounds on polymethacrylic adsorbent. Furthermore, column adsorption experiments were operated to estimate the effect of pore characteristics of the polystyrenic adsorbents on dynamic adsorption behavior, and it is found that both surface area and pore size of the polystyrenic adsorbents significantly affect the dynamic adsorption capacity as well as flow rate.     

A simple procedure for the rapid optimization of reversed-phase separations with ternary mobile phase mixtures

Summary A simple rapid procedure is described for estimating optimum compositions of ternary mobile phase mixtures for the separation of samples by reversed-phase liquid chromatography (RPLC). Retention data in two iso-eluotropic binary mobile phase mixtures (mixtures with equal retention times) are required to initiate the procedure. The logarithm of the capacity factor is assumed to vary linearly with the composition of isoeluotropic ternary mixtures formed by mixing the two limiting binaries. Using the product of resolution factors of adjacent peaks as the criterion, an optimum ternary composition is then calculated. After a chromatogram has been obtained with the predicted optimum ternary mobile phase, the procedure is repeated until no further improvement can be achieved. Examples of the application of the present procedure are described to illustrate its effectiveness.     

Dynamically modified capillary columns for liquid chromatographic separations of biological compounds

Dynamically modified open tubular capillary columns were evaluated for separations of biological and clinical interest. Small diameter, dynamically modified columns were readily prepared by simple addition of cetyltrimethylammonium salts to the mobile phase. Separations similar to those obtained on conventional reversed-phase columns were observed for neutral solutes, and ion-pair reversed-phase separations were observed for charged solutes. Borosilicate and standard flint glasses were evaluated with respect to common etching procedures. A potential clinical application is presented in the separation of antitumor drugs.