Extra-column Effect Research Articles

Research advances in nano liquid chromatography instrumentation

小型化是液相色谱分离技术发展的重要趋势之一,包括仪器外形尺寸的小型化、分离材料粒径的小型化以及色谱柱内径的小型化。色谱柱内径的减小能够降低样品和流动相的消耗,具有更高的质量灵敏度,特别适合用于复杂样品体系的分离分析。纳升液相色谱一般是指使用内径小于100 μm的毛细管色谱柱,流速范围在每分钟几十至几百纳升的色谱技术。由于流速很低,色谱柱体积很小,柱外效应显著,因此对色谱仪器系统各个模块的性能以及系统柱外效应的优化提出了较高的要求。纳升液相色谱的输液装置需要能够准确稳定地输送纳升级流速,具有梯度输液模式,且拥有一定的耐压能力,以适应不同规格的色谱柱类型;进样装置需要能够进行准确重复的进样过程,进样体积及进样方式适合毛细管色谱柱,同时不产生明显的柱外效应;检测装置需要具有较高的灵敏度,且具有较小的柱外扩散;管路与连接系统需要稳定、可靠、易操作,并能够最大限度地减小柱外体积,适配纳升级流速。鉴于目前大多数纳升液相色谱系统与质谱检测器联用,因而本文主要从输液装置、进样装置、管路与连接3个方面对相关技术领域的研究论文、技术专利以及仪器厂商的宣传文件等进行了检索与归纳,综述了这些模块的技术路线与研究进展,同时简要介绍光学吸收型检测装置的优化思路与研究进展,并对部分商品化的纳升液相色谱系统进行了对比。

Reduction of the extra-column band dispersion by a slow transport and splitting of a sample band in isocratic reversed-phase liquid chromatography

Sample introduction method was studied to reduce the extra-column effect in reversed-phase HPLC. Slow transport of a sample band (SToSB) in the pre-column space followed by the introduction of the band into the column at a near-optimum flow rate resulted in larger plate counts for a 1.0 mmID, 5 cm long column as much as 1.4-1.6 times for solutes with a retention factor (k) of 0.5-1.8 compared to a conventional elution method. Further reduction of the extra-column effect was possible by orthogonally splitting the sample band (SplSB) by flow switching during its slow transport followed by the introduction of the leading part of the band into the column. In this case, increased plate counts of up to 2-3 times for solutes with k of 0.5-1.8 were observed for a 1.0 mmID, 5 cm column. The sample introduction method, SToSB in the injector and the pre-column tube of a few μL, was found to reduce the extra-column band variance by 0.4-0.5 μL2 for an UHPLC system with the extra-column volume (Vextra) of ca. 4.6 μL and the system variance (σextra2) of 1.1 μL2 at flow rate of 100 μL/min, while SToSB and subsequent SplSB were found to be more effective, reducing σextra2 by about 0.8 μL2. With an UHPLC instrument with Vextra of about 10 μL and σextra2 of ca. 3.6 μL2 at flow rate of 300 μL/min, 1.4-2.1 times as many plate counts were observed with SToSB and SplSB compared to the normal elution method for early-eluting solutes with k=0.25-1.7 for a column, 2.1 mmID, 5 cm long. With this UHPLC instrument, SToSB and/or SplSB resulted in the reduction of σextra2 by 1.2-2.2 μL2.

Ultrafast Gradient Separation with Narrow Open Tubular Liquid Chromatography.

Separation speed and resolution are two important figures of merit in chromatography. Often, one gains the speed at the cost of the resolution, and vice versa. Scientists have employed short-packed columns for ultrafast separations but encountered challenges such as limited mobile phase velocity, extra-column effect caused band broadening, and column packing difficulty. We have recently demonstrated ultrahigh resolutions of narrow open tubular liquid chromatography (NOTLC); this allows us to trade some of the resolution for speed. In this work, we explored NOTLC for ultrafast LC separations. We used a 2.7 cm (effective length) narrow open tubular (NOT) column and showed a baseline separation of 6 amino acids in less than 700 ms. Ways to further increase the speed were discussed. Using short narrow open tubular (NOT) columns to perform ultrafast separation we overcame the challenges from using short packed columns. To demonstrate the feasibility of using this ultrafast separation technique for practical applications, we separated complex protein digests; peptides were nicely resolved in ∼1 min.

Efficiency of short, small-diameter columns for reversed-phase liquid chromatography under practical operating conditions

Prototype small-size (1.0mm I.D., 5cm long) columns for reversed-phase HPLC were evaluated in relation to instrument requirements. The performance of three types of columns, monolithic silica and particulate silica (2μm, totally porous and 2.6μm, core–shell particles) was studied in the presence of considerable or minimal extra-column effects, while the detector contribution to band broadening was minimized by employing a small size UV-detector cell (6- or 90nL). A micro-LC instrument having small system volume (<1μL) provided extra-column band variance of only 0.01–0.02μL2. The three columns generated about 8500 theoretical plates for solutes with retention factor, k>1–3 (depending on the column), in acetonitrile/water mobile phase (65/35=vol/vol) at 0.05mL/min, with the instrument specified above. The column efficiency was lower by up to 30% than that observed with a 2.1mm I.D. commercial column. The small-size columns also provided 8000–8500 theoretical plates for well retained solutes with a commercial ultrahigh-pressure liquid chromatography (UHPLC) instrument when extra-column contributions were minimized. While a significant extra-column effect was observed for early eluting solutes (k<2–4, depending on column) with methanol/water (20/80=vol/vol) as weak-wash solvent, the use of methanol/water=50/50 as wash solvent affected the column efficiency for most analytes. The results suggest that the band compression effect by the weak-wash solvent associated with partial-loop injection may provide a practical means to reducing the extra-column effect for small-size columns, while the use of an instrument with minimum extra-column effect is highly desirable.

Ternary isocratic mobile phase optimization utilizing resolution Design Space based on retention time and peak width modeling

An optimization procedure of ternary isocratic mobile phase composition in the HPLC method using a statistical prediction model and visualization technique is described. In this report, two prediction models were first evaluated to obtain reliable prediction results. The retention time prediction model was constructed by modification from past respectable knowledge of retention modeling against ternary solvent strength changes. An excellent correlation between observed and predicted retention time was given in various kinds of pharmaceutical compounds by the multiple regression modeling of solvent strength parameters. The peak width of half height prediction model employed polynomial fitting of the retention time, because a linear relationship between the peak width of half height and the retention time was not obtained even after taking into account the contribution of the extra-column effect based on a moment method. Accurate prediction results were able to be obtained by such model, showing mostly over 0.99 value of correlation coefficient between observed and predicted peak width of half height. Then, a procedure to visualize a resolution Design Space was tried as the secondary challenge. An artificial neural network method was performed to link directly between ternary solvent strength parameters and predicted resolution, which were determined by accurate prediction results of retention time and a peak width of half height, and to visualize appropriate ternary mobile phase compositions as a range of resolution over 1.5 on the contour profile. By using mixtures of similar pharmaceutical compounds in case studies, we verified a possibility of prediction to find the optimal range of condition. Observed chromatographic results on the optimal condition mostly matched with the prediction and the average of difference between observed and predicted resolution were approximately 0.3. This means that enough accuracy for prediction could be achieved by the proposed procedure. Consequently, the procedure to search the optimal range of ternary solvent strength achieving an appropriate separation is provided by using the resolution Design Space based on accurate prediction.

Practical comparison of LC columns packed with different superficially porous particles for the separation of small molecules and medium size natural products

Commercial C(18) columns packed with superficially porous particles of different sizes and shell thicknesses (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes. Significant efficiency loss was observed on 2.1-mm id columns even with a low dispersion ultra-high pressure liquid chromatography system. The Kinetex 4.6-mm id column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes. The Kinetex 2.1-mm id column packed with 1.7-μm particles did not deliver the expected performance and the possible reasons besides extra column effect have been proved to be frictional heating effect and poor column packing quality. Different column retentivities and selectivities have been observed on the four C(18) columns of different brands for the natural product separation. Column batch-to-batch variability that has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column.

Polydimethylsiloxane Connection for Quartz Microchips in a High-Pressure System

An interconnecting technique, the "PDMS connection method", for quartz microchips in a high-pressure system is presented. The connection between quartz microchips is an essential technology for modular microfluidic devices, such as microchip-HPLC. PDMS was applied to the seal material, being spread on the seal side of the chips, and set into the metal housing. The characteristics of the PDMS connection method concerning pressure resistance and the extension of the peak were examined. The experimental results showed a good seal at 5 MPa, which seem to be sufficient for realizing microchip-HPLC utilizing a monolithic silica capillary column as a separation medium. The influence of the extra column effect on chromatographic separation was almost the same as in the case using a commercial union fitting. In addition, the PDMS connection enabled the detachability of chip-based modules with user-friendliness. Our experimental findings suggest that the novel PDMS connection method can possibly be applied as a generic technology in high-pressure microTAS.

The Study of the Submicroparticle Packed Column

In this paper, a new type of high performance packed column, the submicroparticle packed column (SPPC), has been proposed. SPPC has the advantages of a microparticle packed column, high efficiency and high analytical speed, and a classical packed column, high column capacity, low cost, and easy operation. In some respects, its properties are similar to that of a capillary, which can analyze high boiling point components or wide boiling range samples at a relatively low column temperature. The height equivalent to an effective plate can be as low as 0.155 mm, and the plate number per second is 95. Moreover, the extra-column effect of the chromatographic system and its diminutiveness and the influences on column efficiency (such as average carrier gas velocity, make-up gas flow, and temperature) are also discussed in detail. SPPC is suitable for fast and trace analysis in many fields.

Isotopic Backmixing in a Large-Scale Enrichment Column in a Chemical Enrichment Method

The Asahi chemical enrichment pilot plant with four large-scale 1-mm-diam enrichment columns has been operated using the «super» process since June 1987. Uranium with 3.3% enrichment was recovered in April 1988, and higher efficiencies have been observed in the pilot plant than in the bench-scale plant, which has 0.1-mm-diam enrichment columns. A possible reason is that the isotopic backmixing in the pilot plant is much smaller than in the bench-scale plant.Quantitative and statistical studies imply that both the extracolumn volume ratio and wall effect contribute to the smaller backmixing

Influence of the eluent composition on column efficiency in reversed-phase high-performance liquid chromatography

The dependence of column efficiency on the eluent (MeOH/H2O) composition in a reversed-phase liquid chromatography system within a wide concentration range has been systematically examined. It is shown that when the intracolumn effect of mass transfer and diffusion is the main factor controlling band broadening the column efficiency decreases with the increase of the viscosity of the MeOH/H2O mixture; on the other hand, when the extra-column effect is the main factor an increase in the viscosity of the eluents will help in improving column efficiency. The column efficiency is also related to the properties of the sample.

Extra-column effects in high-performance liquid chromatography : I. Theoretical study of the injection problem

The effect of the injection process on the column efficiency depends on the injection time, the injection volume and the technique of sampling (syringe on-line or stop-flow injection through a septum-type injector port or valve injection in either the normal or the split mode). The injection process can be described by a contribution to the plate height that is proportional to the square of the length of the solute zone at the column inlet (Δ x) and iversely proportional to the square of the column length and to a constant describing the concentration profile in the solute zone. A simple model based on the combined effect of solute displacement due to the injection flow and to the mobile phase flow makes possible the calculation of Δ. The effects of injection time and volume and volume are complex and depend on the sampling system and on geometrical parameters such as the column internal cross-sectional area, the sampling canal intenal and external cross-sectional areas and two co- efficients related to the flow pattern at the column inlet. In some conditions, the model proposed predicts, in agreement with experimental results, a non-linear dependence of the plate height on the square of the injection volume. Finally, the influence of particle size on the extra-column effect due to injection depends cosely on the experimental conditions: the results are different when the column is operated at maximal efficiency and when we want to achieve a given number of theoretical plates in a given time. In some instances the injection effect is independent on the particle size and in others it is inversely proportional to d p 4 .

Automatic Analysis for Sugars by Liquid Chromatography

A quantitative determination by a simple detection system for carbohydrates with elution chromatography was developed. This method depends upon the detection of ultraviolet absorbing chromophere, with absorption maximum in the 260-270 mμ region, produced by heating the carbohydrates in the eluent solution without reagent. In the product from fructose, the existence of furfural and formic acid was confirmed . The heating condition and eluant buffer (conc., pH) affected the intensity of ultraviolet absorption produced. Under the condition of heating below 190°C and shorter than 20 min, the greater efficiency was obtained at higher temperature and longer period . Higher concentration and neutral pH of borate buffer were more effective . The molar absorption coefficient at 260 mp of mono- and oligosaccharides heated in a solution of 0.8 M borate buffer pH 7.0 for 9.5 min at 190°C was 500-3000 . The coefficient of variation of this method was less than 3.5%. A heating period was obtained by adjusting the diameter and length of teflon tube connected with the chromatographic column. In order to decrease the extra column effect, it was effective to decrease the diameter of tube (0.5 mm) and heating period, and increase the heating temperature . Several examples for mono- and oligosaccharides and oligodextrines were shown.